Single lentiviral vector system for induced pluripotent (ips) stem cells derivation

ABSTRACT

The present invention is based on the discovery that a single lentiviral vector expressing multiple individual transcription factor proteins from a single multi-cistronic mRNA can reprogram a fibroblast cell to a stem cell-like cell. These reprogrammed induced pluripotent stem (iPS) cells are pluripotent. Additions of the Cre-LoxP sequences into the single lentiviral vector facilitate excision of the vector after reprogramming in achieved. Addition of a maker gene into the single lentiviral vector facilitates detection of the presence of the vector in an iPS. The invention provides compositions and methods of producing iPS cells using a single multi-cistronic lentiviral vector.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of co-pending U.S.application Ser. No. 13/080,728 filed on Apr. 6, 2011, which is acontinuation application of the International Application No.PCT/US2009/059660, filed Oct. 6, 2009, which designates the UnitedStates, which claims benefit under 35 U.S.C. §119(e) of the U.S.Provisional Application No. 61/103,091 filed on Oct. 6, 2008, thecontents of which are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 5, 2014, isnamed 701586-063733-C2_SL.txt and is 87,936 bytes in size.

BACKGROUND OF INVENTION

The capacity of embryonic stem cells (ESCs) to give rise to all types ofsomatic cells together with their ability to grow indefinitely inculture underscores their potential for in vivo therapeuticapplications. However, many challenges exist, for example, ES cells arenot genetically identical to the organism from which they are harvested,and thus rejection and immunogenicity are two concerns that potentiallylimit their future use in clinical transplantation. In addition, ethicalconcerns have been raised regarding the derivation of human ES cellsfrom human embryos. For these reasons considerable effort has beeninvested in attempting to derive pluripotent stem cells from post-nataltissue that may be employed for isogenic or autologous transplantation.However, the generation of patient-specific autologous ESCs istechnically challenging and further complicated by ethical concerns,significantly limiting their potential for clinical transplantation. Thereprogramming of fibroblasts to an ESC-like state, pioneered by Yamanakaand colleagues, has advanced stem cell research (Takahashi and Yamanaka,2006, Cell 126:663-676) by circumventing these obstacles. These socalled ‘induced Pluripotent Stem (iPS) cells’ derived from mouse orhuman fibroblasts have demonstrated that an entire organism can bederived from readily accessible post-natal somatic cells. iPS cellsprovide a powerful in vitro model system for the study of the molecularmechanisms of reprogramming and have been successfully employed inproof-of-principle cell-based therapies in mouse models of disease.However, to date the derivation of iPS cells has required multipleindividual viral vectors to deliver the constellation of transcriptionfactors (typically OCT4, SOX2, KLF4, and c-MYC) required to inducereprogramming. The application of sufficient quantities of each virusneeded to deliver four factors simultaneously to each target cellresults in high numbers of genomic integrations in successfullyreprogrammed progeny. This presence of multiple viral integrationsacross the genome makes their genetic elimination to produce safer iPScells very difficult. Moreover, many cells will receive only one, two orthree factors, making it difficult to study the biochemistry ofreprogramming on a homogeneous population of cells. Hence there is aneed for improved methods that provide consistent delivery ofreprogramming transcription factors and with minimal or no viralintegrations across the genome to produce safer iPS cells.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that a single lentiviralvector expressing multiple individual transcription factor proteins froma single multi-cistronic mRNA can reprogram a fibroblast cell to a stemcell-like cell. These reprogrammed iPS cells are pluripotent asevidenced by their ability to divide unlimited times to form teratomasand to contribute to chimeric embryos. The integration of a single copyof the lentiviral vector was sufficient to reprogram the fibroblastcell.

Furthermore, the inventors demonstrate efficient derivation oftransgene-free iPS cells using an excisable polycistronic lentiviralvector. In other words, after the integration of the polycistroniclentiviral vector that brought about the deprogramming of differentiatedcells, the integrated polycistronic lentiviral vector can be efficientlyexcised to leave behind lentivirus-free iPS cells. A direct comparisonof iPS cell clones before and after excision reveals that removal of thereprogramming vector markedly improves the developmental potential ofiPS cells and significantly augments their capacity to undergo directeddifferentiation in vitro.

Furthermore, a specific marker gene can be incorporated into thepolycistronic lentiviral vector for the purpose of monitoring thepresence or absence of the lentivirus in the lentivirus-integrated iPScells or lentivirus-free iPS cells respectively. The specific markergene can be one that expresses optically visible proteins such as thegreen fluorescent protein or the red “cherry” fluorescent proteindescribed herein or an enzyme whose activity can be assayed, e.g.,thymidine kinase.

Accordingly, in one embodiment, the invention described herein providesa lentiviral vector particle capable for reprogramming a somatic cell toa stem-cell-like cell, the vector particle comprising a nucleic acidsequence comprising encoding sequences of multiple individualtranscription factor proteins sufficient to reprogram a somatic cell.For example, a sequence encoding: (a) a Oct4 gene; (b) a Klf4 gene; (c)a Sox2 gene; (d) a c-Myc gene; (e) a first ‘self-cleaving’ 2A peptide;(f) a second ‘self-cleaving’ 2A peptide; (g) an internal ribosome entrysite (IRES); wherein the nucleic acid sequence is operably linked to apromoter; and wherein the sequences encoding OCT4, KLF4, SOX2, c-MYC,first and second ‘self-cleaving’ 2A peptides, and the IRES aretranscribed from the promoter as a multi-cistronic RNA.

In another embodiment, the sequence encoding: (a) a Oct4 gene; (b) aKlf4 gene; (c) a Sox2 gene; (d) a specific marker gene; (e) a first‘self-cleaving’ 2A peptide; (f) a second ‘self-cleaving’ 2A peptide; (g)an internal ribosome entry site (IRES); wherein the nucleic acidsequence is operably linked to a promoter; and wherein the sequencesencoding OCT4, KLF4, SOX2, c-MYC, first and second ‘self-cleaving’ 2Apeptides, and the IRES are transcribed from the promoter as amulti-cistronic RNA, wherein the specific marker gene encodes afluorescent protein or an enzyme, e.g. thymidine kinase.

In one embodiment, the nucleic acid sequence comprising encodingsequences of multiple individual transcription factor proteinssufficient to reprogram a somatic cell can be selected from the groupconsisting of OCT4, KLF4, SOX2, LIN28, NANOG and c-MYC. In someembodiments, the nucleic acid sequence comprises encoding sequences ofmultiple individual transcription factor proteins sufficient toreprogram a somatic cell comprises three or four of the transcriptionfactors selected from the group consisting of OCT4, KLF4, SOX2, LIN28,NANOG and c-MYC. For example, the nucleic acid sequence comprisesencoding sequences of OCT4, KLF4 and SOX2, sequences of OCT4, KLF4,LIN28 and NANOG, sequences of OCT4, LIN28, NANOG and c-MYC.

In one embodiment, the somatic cell is a mammalian cell. In oneembodiment, the somatic cell is a mammalian cell derived from internalorgans-heart, kidney, liver, lungs, bladder, intestines; skin, bone,blood, cartilage and connective tissues.

In one embodiment, the sequences encoding the multiple individualtranscription factors such as the four genes: Oct4, Klf4, Sox2, andc-Myc, are arranged in tandem, wherein the genes are oriented in thesense direction, and wherein the genes are arranged in any order.

In some embodiments, the sequence encodes only four genes selected fromthe group consisting of Oct4, Klf4, Sox2, Lin28, Nanog and c-Myc. Inother embodiments, sequence encodes only three genes selected from thegroup consisting of Oct4, Klf4, Sox2, Lin28, Nanog and c-Myc, e.g. Oct4,Klf4, and Sox2; Oct4, Klf4, and c-Myc; or Oct4, Sox2 and Lin28. In theembodiments where only three genes are used, the specific marker genecan form the fourth gene in the tandemly arranged sequence. For example,Oct4, Klf4, Sox2 and a specific marker gene, Oct4, Klf4, Lin28 and aspecific marker gene, and Oct4, Lin28, c-Myc and a specific marker gene.

In one embodiment, the sequence encoding internal ribosome entry site(IRES) is between the second and third genes in the tandem arrangementof the four genes, e.g. Oct4, Klf4, Sox2, and c-Myc. The arrangement isthen Oct4, Klf4, IRES, Sox2, and c-Myc.

In one embodiment, the sequence encoding the first ‘self-cleaving’ 2Apeptide is between the first and second genes in the tandem arrangementthe four genes, e.g. Oct4, Klf4, Sox2, and c-Myc. The arrangement isthen Oct4, 1^(st) 2A, Klf4, IRES, Sox2, and c-Myc.

In one embodiment, the sequence encoding the second ‘self-cleaving’ 2Apeptide is between the third and forth genes in the tandem arrangementthe four genes, e.g. Oct4, Klf4, Sox2, and c-Myc. The arrangement isthen Oct4, first ‘self-cleaving’ 2A peptide, Klf4, IRES, Sox2, second‘self-cleaving’ 2A peptide, and c-Myc.

In some embodiments, the first and the second ‘self-cleaving’ 2A peptideis selected from the group consisting of F2A, E2A, T2A and P2A, whereinthe second ‘self-cleaving’ 2A peptide is different from the first‘self-cleaving’ 2A peptide.

In one embodiment, the promoter is inducible, for example, atetracycline regulated promoter.

In another embodiment, the promoter is constitutive, for example, aEF-1alpha promoter

In one embodiment, the polycistronic lentiviral vector comprisessequences that facilitate the excision of the integrated vector, e.g.Cre-LoxP and Cre-ERT2 sequences wherein the excision is executed withCre recombinase protein or tamoxifen via the inducible Cre-ERT2recombinase respectively.

In another embodiment, the invention described herein provides a methodof reprogramming a somatic cell; the method comprising contacting asomatic cell with a lentiviral vector described herein. In oneembodiment, the somatic cell is a mammalian cell. In one embodiment, thesomatic cell is a mammalian cell derived from internal organs-heart,kidney, liver, lungs, bladder, intestines; skin, bones, blood, cartilageand connective tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the schematic representation of pHAGE-STEMCCATetO/minmCMV, inducible version) generation of a single lentiviralvector expressing a stem-cell cassette. The engineered stem cellcassette consists of a single multicistronic mRNA transcribed under thecontrol of a doxycycline-inducible TetO-miniCMV promoter. The mRNAcontains an IRES element separating two fusion cistrons. The twocistrons consist of Oct4 and Sox2 coding sequences fused to Klf4 andc-Myc, respectively, through the use of intervening sequences encoding‘self-cleaving’ 2A peptides (F2A and E2A). LTR: long terminal repeat;PSI: packaging signal; RRE: rev responsive element; cpPu: centralpolypuryne tract; WPRE: Woodchuck hepatitis virus post-transcriptionalregulatory element.

FIG. 1B shows the Western blot analysis of lysates from 293T transfectedcells. Cells were transfected with pHAGE-Tet-STEMCCA and maintained indoxycycline for 72 hr before lysate preparation. Cells transfected witheither mock vectors or four monocistronic pHAGE vectors encoding thefour individual transcription factors were used as negative (−) orpositive (+) controls, respectively.

FIG. 2A shows representative pictures of iPS cells derived using theconstitutive pHAGE-EF1α-STEMCCA: the colony morphology (Phase), highalkaline phosphatase activity (Alk Phos), SSEA1 immunostaining, andSox2-GFP reporter gene expression.

FIG. 2B shows representative pictures of iPS cells derived using theinducible pHAGE-Tet-STEMCCA vector: the colony morphology (Phase), highalkaline phosphatase activity (Alk Phos), SSEA1 immunostaining, andSox2-GFP reporter gene expression.

FIG. 2C shows the expression of ES cell ‘marker’ genes detected byRT-PCR in four representative iPS cell clones generated by theconstitutive (EF1α) or inducible (Tet) STEMCCA vector. Nat1 is aconstitutively expressed gene and serves as a control for loading.Representative samples from unmanipulated mouse embryonic fibroblasts(MEF) and mouse ES cells are also shown. An iPS cell sample preparedwithout RT was used as negative control (−RT).

FIG. 2D shows the Southern blot analysis of genomic DNA (gDNA) purifiedfrom 6 representative iPS clones produced with the constitutive (EF1α)or inducible (tet) vector. gDNA was digested with BglII to obtain a bandof 6.7 Kb in the EF1α colonies or 8.3 Kb in the Tet colonies,representing most of the proviral genome. For control, pHAGE-Tet-STEMCCAplasmid DNA representing 1 or 2.5 copies of the insert was digested withBglII. A single band of the expected size of the proviral gene insertionis present in all clones. The density of each band indicates between 1-3proviral integrations in each clone.

FIG. 3 shows the dynamics of reprogramming using a single lentiviralstem-cell cassette. Analysis of GFP expression over time in TTFspurified from Sox2-GFP M2rtTA double knock-in mice infected withpHAGE-Tet-STEMCCA vector. Transduced cells from independent wells werecollected at each time point following doxycycline exposure. GFPexpression was analyzed using a FACScan machine. The Day 5 result wasindistinguishable from background GFP expression.

FIG. 4A shows the teratomas derived from iPS lines produced withpHAGE-EF1α-STEMCCA vector, showing differentiation into cell types ofall three germ layers: endoderm (end), mesoderm (mes) and ectoderm(ect). Images are representative of two independent experiments testingthree individual iPS clones for each construct.

FIG. 4B shows the teratomas derived from iPS lines produced withpHAGE-Tet-STEMCCA vector, showing differentiation into cell types of allthree germ layers: endoderm (end), mesoderm (mes) and ectoderm (ect).Images are representative of two independent experiments testing threeindividual iPS clones for each construct.

FIG. 4C shows the iPS cells generated with pHAGE-Tet-STEMCCA vector fromTTFs of a Sox2-GFP Rosa26-M2rtTA mouse show high levels of embryoniccontribution following injections into blastocysts. Chimerism isevidenced by Sox2-GFP expression in neural crest-derived tissues in 9 of12 mid-term embryos.

FIG. 5: STEMCCA transgene expression in transduced fibroblasts and inestablished iPS cell lines. Expression of the multicistronic STEMCCAtranscript in transduced tail tip fibroblasts (TTFs) and established iPSclones was assessed by semi-quantitative RT-PCR. Estimated expressionlevels of the STEMCCA transcript relative to expression of the controlconstitutively expressed gene, Nat1 is shown for each sample. Ten daysafter doxycycline (dox) exposure, TTFs transduced with pHAGE-Tet-STEMCCAlentivirus (d10 infected TTFs) express easily detected levels of theSTEMCCA transcript. In contrast, following dox withdrawal, anestablished iPS clone (Tet STEMCCA iPS) shows significant downregulationof expression. An established iPS clone generated using the‘constitutive’ pHAGE-EF1a-STEMCCA lentivirus (EF1a STEMCCA iPS),expresses the STEMCCA transcript at lower levels than the dox-exposedTTFs, but at higher levels than the Tet STEMCCA iPS clone. Error barsindicate standard deviations (n=3).

FIG. 6A is a map of pHAGE-Tet-STEMCCA gene transfer plasmid showing therestriction enzyme sites. The transfer plasmid is also known aspHAGE2-TetOminiCV-Oct4F2aKlf4-IRES-Sox2E2AcMyc-W.

FIG. 6B is a map of pHAGE-Tet-STEMCCA gene transfer plasmid showing themajor operational elements of the plasmid. The transfer plasmid is alsoknown as pHAGE2-TetOminiCV-Oct4F2aKlf4-IRES-Sox2E2AcMyc-W.

FIG. 7A is a map of pHAGE-EF1α-STEMCCA gene transfer plasmid showing therestriction enzyme sites. The transfer plasmid is also known aspHAGE2-EF1αFull-Oct4F2aKlf4-IRES-Sox2E2AcMyc-W.

FIG. 7B is a map of pHAGE-EF1α-STEMCCA gene transfer plasmid showing themajor operational elements of the plasmid. The transfer plasmid is alsoknown as pHAGE2-EF1αFull-Oct4F2 aKlf4-IRES-Sox2E2AcMyc-W.

FIG. 8A shows the schematic representation of the STEMCCA-loxP (SEFL) orSTEMCCA-loxP-RedLight (SEFCL) lentiviral vector, excisable singlelentiviral vectors for the generation of iPS cells free of exogenoustransgenes. The ‘RedLight’ indicate the mCherry gene in the vector. Thisvector is a constitutive promoter EF1α.

FIG. 8B shows the analysis of iPS cells created using theSTEMCCA-loxP-RedLight vector using flow cytometry to detect mCherryfluorescence. Cells before and after Cre treatments are shown.

FIG. 8C shows the Southern blot analysis of genomic DNA (gDNA) purifiedfrom two representative iPS clones produced with the STEMCCA-loxPvector, before and after Cre-mediated excision.

FIG. 8D shows the expression of the STEMCCA transcript was analyzed byRT-PCR to confirm excision. As expected, clones SEFL1-Cre and SEFL2-Creshowed no detectable STEMCCA transcript. Nat1 is a constitutivelyexpressed gene and serves as a control for loading.

FIG. 9A shows representative images of iPS cells derived using theconstitutive STEMCCA-loxP vector before and after Cre-mediated excisionshowing normal colony morphology (Phase), Sox2-GFP reporter geneexpression, SSEA1 positive immunostaining, and robust alkalinephosphatase activity (Alk Phos).

FIG. 9B shows the expression of ESC ‘marker’ genes detected by RT-PCR iniPS cell clones before (SEFL1 and SEFL2) and after excision (SEFL1-Creand SEFL2-Cre). Representative samples from murine ESC and unmanipulatedtail tip fibroblasts (TTFs) are also shown for comparison. An iPS cellsample prepared without RT was used as negative control (−RT).

FIG. 9C shows the analysis of the promoter regions of Nanog and Oct4genes by determining the methylation status using bisulfite sequencing.Similar to ESC, all iPS cell clones (SEFL1, SEFL1-Cre, SEFL2 andSEFL2-Cre) showed mostly unmethylated CpG motifs (open circles) in sharpcontrast to parental TTFs, in which the extracted DNA was mostlymethylated (closed circles).

FIG. 10A shows the teratomas derived from iPS cell lines produced withSTEMCCA-loxP vector, before and after Cre excision, showingdifferentiation into cell types of all three germ layers: endoderm(end), mesoderm (mes) and ectoderm (ect).

FIG. 10B shows representative images of embryos with iPS cells generatedwith the constitutive STEMCCA vector (no Cre-excision) displayed highlevels of embryonic contribution following injection into blastocysts,but also induced gross morphological abnormalities

FIG. 10C shows representative images of embryos with iPS cells generatedusing the STEMCCA-loxP vector with subsequent Cre excision produces ahigher percentage of chimeric embryos with normal developmentalmorphology. Chimerism is evidenced by Sox2-GFP expression in neuralcrest-derived tissues.

FIG. 10D shows the derivation of neonatal chimeric mice from blastocystsinjected with iPS cells generated using the STEMCCA-loxp vector withsubsequent Cre excision. Chimerism is evidenced by dark coat color.

FIG. 11A shows representative RT-PCR data of iPS clones subjected to invitro differentiation toward primitive streak/endoderm using activin Astimulation (Act A).

FIG. 11B shows the quantitative RT-PCR data for iPS cells stimulated invitro using activin A. Data is expressed as fold change normalized to18S expression. mRNA extracted on day 0 (light grey columns) or day 5(dark grey columns) of activin A stimulation served as the template forqRT-PCR.

FIG. 12 shows the Southern blot analysis of iPS cell clones generatedwith STEMCCA-loxP (SEFL) or STEMCCA-loxP-RedLight (SEFCL) showing numberof proviral integrations. Several clones displaying a single integrationare shown (asterisk). gDNA was digested with BamHI that cuts once withinthe provirus. Blots were probed against the WPRE element present in bothSTEMCCA vectors.

FIG. 13 shows that the PCR of a c-Myc to WPRE fragment using gDNAisolated from several iPS sub-clones post Cre excision (SEFL1-Cre andSEFL2-Cre) shows absence of band. gDNA samples isolated from theparental lines (SEFL1 and SEFL2) show positive PCR amplification. gDNAisolated from a previously generated iPS clone containing a single copyof the constitutive STEMCCA was used as positive control for PCRreaction (Control).

FIG. 14 shows that the quantitative real time PCR (qRT-PCR) performed ontwo independent iPS clones generated with the STEMCCA-loxP vector (SEFL1and SEFL2) produces equivalent levels of STEMCCA expression. The STEMCCAtranscript was not present after Cre-mediated excision of the STEMCCAvector (SEFL1 Cre and SEFL2-Cre). N.D.: Not Detected.

FIG. 15A is a map of pHAGE-EF1αFull-STEMCCA-W-RedLight-LoxP genetransfer plasmid showing the restriction enzyme sites. This vector hasthe LoxP flanking the STEMCCA and the fourth gene in the cassette,c-Myc, has been replaced with a marker gene, mCherry, which codes for ared fluorescent protein. This transfer plasmid is also known aspHAGE-EF1α-STEMCCA-LoxP-RedLight andpHAGE2-EF1αFull-Oct4F2aKlf4-IRES-Sox2E2AmCherry-W-LoxP.

FIG. 15B is a map of pHAGE-EF1αfull-STEMCCA W-RedLight-LoxP genetransfer plasmid showing the major operational elements of the plasmid.This vector has the LoxP flanking the STEMCCA and the fourth gene in thecassette, c-Myc, has been replaced with a marker gene, mCherry, whichcodes for a red fluorescent protein. This transfer plasmid is also knownas pHAGE-EF1α-STEMCCA-LoxP-RedLight andpHAGE2-EF1αFull-Oct4F2aKlf4-IRES-Sox2E2AmCherry-W-LoxP.

DETAILED DESCRIPTION OF THE INVENTION Definitions of Terms

As used herein, the term “a somatic cell” refers to any cell forming thebody of an organism that are not germline cells (e.g. sperm and ova, thecells from which they are made (gametocytes)) and undifferentiated stemcells. Internal organs, skin, bones, blood and connective tissue are allmade up of somatic cells.

As used herein, the term “self-cleaving 2A peptide” refers to relativelyshort peptides (of the order of 20 amino acids long, depending on thevirus of origin) containing the consensus motif D-V/I-E-X-N-P-G-P (SEQID NO: 52; preferred embodiments disclosed as SEQ ID NOS 1-2). They wereoriginally thought to mediate the autocatalytic proteolysis of the largepolyprotein, but are now understood to act co-translationally, bypreventing the formation of a normal peptide bond between the glycineand last proline, resulting in the ribosome skipping to the next codon,and the nascent peptide cleaving between the Gly and Pro. Aftercleavage, the short 2A peptide remains fused to the C-terminus of the‘upstream’ protein, while the proline is added to the N-terminus of the‘downstream’ protein. The 2A peptide was identified among Picornavirusesbut in a different sub-group, the Aphthoviruses, a typical example ofwhich is the Foot-and-mouth disease virus.

As used herein, the term “promoter” refers to a regulatory region of DNAgenerally located upstream (towards the 5′ region of the sense strand)of a gene that allows transcription of the gene. The promoter containsspecific DNA sequences and response elements that are recognized byproteins known as transcription factors. These factors bind to thepromoter sequences, recruiting RNA polymerase, the enzyme thatsynthesizes the RNA from the coding region of the gene.

As used herein, the term “cistron” refers to a section of the DNAmolecule that specifies the formation of one polypeptide chain, i.e.coding for one polypeptide chain. A fusion cistron refers to two or moresections of different DNA molecule fused together to specify theformation of one polypeptide chain.

As used herein, the term “multi-cistronic RNA” or “multi-cistron RNA”refers to an RNA that contains the genetic information to translate toseveral proteins. In contrast, a monocistronic RNA contains the geneticinformation to translate only a single protein. In the context of thepresent invention, the multi-cistronic RNA transcribed from thelentivirus in the Example 1 is translated to four proteins: OCT4, KLF4,SOX2, and c-MYC. Likewise, the multi-cistronic RNA transcribed from thelentivirus in the Example 2 is translated to four proteins: OCT4, KLF4,SOX2, and mCHERRY fluorescent protein.

As used herein, the term “arranged in tandem” refers to the arrangementof the genes back to back, one following or behind the other, in asingle file on a nucleic acid sequence. The genes are ligated togetherback to back in a single file on a nucleic acid sequence, with thecoding strands (sense strands) of each gene ligated together on anucleic acid sequence.

As used herein, the term “sense strand” refers to the DNA strand of agene that is translated or translatable into protein. When a gene isoriented in the “sense direction” with respect to the promoter in anucleic acid sequence, the “sense strand” is located at the 5′enddownstream of the promoter, with the first codon of the protein isproximal to the promoter and the last codon is distal from the promoter.

The term “constitutive” use herein refers to “all the time” orconstantly. For example, a gene product that is expressed all the timeis constitutively expressed. A “constitutive” promoter is active all thetime, transcribing the attached gene to primary RNA transcript all thetime. Such a promoter is unregulated and it allows for continualtranscription of its associated gene. Examples of “constitutive”eukaryotic promoters are elongation factor 1 alpha (EF1α) andcytomegalovirus (CMV).

As used herein, the term “inducible” refers to regulatable. For example,the activity of an inducible promoter can be turned on or off, i.e.regulated by the presence or absence of biotic or abiotic factors.Examples of inducible promoters include: chemically-regulated promoters,including promoters whose transcriptional activity is regulated by thepresence or absence of alcohol, tetracycline, steroids, metal and othercompounds; and physically-regulated promoters, including promoters whosetranscriptional activity is regulated by the presence or absence oflight and low or high temperatures. Examples chemically induciblepromoters can have hormone-responsive elements (HREs), metal-responsiveelements (MREs), heat shock-responsive elements (HSREs), tetracyclineoperator sequence (TetO) and interferon-responsive elements (IREs).

As used herein the term “comprising” or “comprises” is used in referenceto vector particles, vector systems, methods, and respectivecomponent(s) thereof, that are essential to the invention, yet open tothe inclusion of unspecified elements, whether essential or not.

The term “consisting of” refers to vector particles, vector systems,methods, and respective components thereof as described herein, whichare exclusive of any element not recited in that description of theembodiment.

As used herein, the term “exogenous copy” of a gene refers to thenon-genomic copy of a gene, an added copy of gene that is introducedinto the cell, for example, in the form of a cDNA copy. The term“endogenous” use herein means the original copy of the gene found in thegenome of the cell.

As used herein, the term “transgene” refers to a nucleic acid sequencewhich is partly or entirely heterologous, i.e., foreign, to thetransgenic animal or cell into which it is introduced, or, is homologousto an endogenous gene of the transgenic animal or cell into which it isintroduced, but which is designed to be inserted, or is inserted, intothe animal's genome in such a way as to alter the genome of the cellinto which it is inserted (e.g., it is inserted at a location whichdiffers from that of the natural gene or its insertion results in aknockout). A transgene can be operably linked to one or moretranscriptional regulatory sequences and any other nucleic acid, such asintrons, that may be necessary for optimal expression of a selectednucleic acid.

“Functional variant” refers to a nucleic acid or protein having anucleotide sequence or amino acid sequence, respectively, that is“identical,” “essentially identical,” “substantially identical,”“homologous” or “similar” to a reference sequence which can, by way ofnon-limiting example, be the sequence of an isolated nucleic acid orprotein, or a consensus sequence derived by comparison of two or morerelated nucleic acids or proteins, or a group of isoforms of a givennucleic acid or protein. Non-limiting examples of types of isoformsinclude isoforms of differing molecular weight that result from, e.g.,alternate RNA splicing or proteolytic cleavage; and isoforms havingdifferent post-translational modifications, such as glycosylation; andthe likes.

As used herein, the term “variants” or “variant” refers to a nucleicacid or polypeptide differing from a reference nucleic acid orpolypeptide, but retaining essential properties thereof. Generally,variants are overall closely similar, and, in many regions, identical tothe reference nucleic acid or polypeptide. Thus “variant” forms of atranscription factor are overall closely similar, and capable of bindingDNA and activate gene transcription.

As used herein, the term “conservative amino acid substitution” is onein which the amino acid residue is replaced with an amino acid residuehaving a side chain with a similar charge and size. Families of aminoacid residues having side chains with similar charges have been definedin the art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

As used herein, the term “stem cell-like cell” refers to refers to acell that has been transfected with exogenous gene copies of multipleindividual transcription factors such as OCT4, KLF4, SOX2, C-MYC, LIN28and NANOG and the cell has acquired characteristics of an unspecialized“stem cell” such as the ability to self-renewal and potency. The potencycan be pluripotent or multipotent. A stem cell-like cell is lessdifferentiated than its oricinal cell prior to the transfection with theexogenous genes described herein.

As used herein, the term “stem cell” refers to a cell that has theability to self-renewal, i.e., to go through numerous cycles of celldivision while maintaining the undifferentiated state, and has potency,i.e. the capacity to differentiate into specialized cell types, e.g. anerve cell or a skin cell.

As used herein, the term “pluripotent” refers to the potential of a stemcell to make any differentiated cell in the body but not those of theplacenta which is derived from the trophoblast.

As used herein, the term “multipotent” refers to the ability to onlydifferentiate into a limited number of types. For example, the bonemarrow contains multipotent stem cells that give rise to all the cellsof the blood but not to other types of cells. Multipotent stem cells arefound in adult animals; perhaps most organs in the body (e.g., brain,liver) contain them where they can replace dead or damaged cells. Theseadult stem cells may also be the cells that—when one accumulatessufficient mutations—produce a clone of cancer cells.

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle, encodes at leastan exogenous nucleic acid. The vector and/or particle can be utilizedfor the purpose of transferring any nucleic acids into cells either invitro or in vivo. Numerous forms of viral vectors are known in the art.The term virion is used to refer to a single infective viral particle.“Viral vector”, “viral vector particle” and “viral particle” also referto a complete virus particle with its DNA or RNA core and protein coatas it exists outside the cell.

The term “replication incompetent” as used herein means the viral vectorcannot further replicate and package its genomes. For example, when thecells of a subject are infected with replication incompetent recombinantlentivirus such as the human immunodeficiency virus (HIV) or felineimmunodeficiency virus (FIV), the heterologous (also known as transgene)gene is expressed in the patient's cells, but, the rHIV is replicationdefective (e.g., lacks essential packaging elements of the virus) andviral particles cannot be formed in the patient's cells.

The term “gene” means the nucleic acid sequence which is transcribed(DNA) and translated (mRNA) into a polypeptide in vitro or in vivo whenoperably linked to appropriate regulatory sequences. The gene may or maynot include regions preceding and following the coding region, e.g. 5′untranslated (5′UTR) or “leader” sequences and 3′ UTR or “trailer”sequences, as well as intervening sequences (introns) between individualcoding segments (exons).

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof (“polynucleotides”) in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid molecule/polynucleotide also implicitly encompasses conservativelymodified variants thereof (e.g. degenerate codon substitutions) andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka etal., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell.Probes 8: 91-98 (1994)). Nucleotides are indicated by their bases by thefollowing standard abbreviations: adenine (A), cytosine (C), thymine(T), and guanine (G).

As used herein, the term “operably linked” refers to that the regulatoryelements in the nucleic acid construct are in-frame with a nucleic acidcoding for a protein or peptide.

Embodiments of the present invention is based on the discovery that asingle lentiviral cassette can be used to create a single lentiviralvector, expressing four individual transcription factor proteins from asingle multi-cistronic mRNA to reprogram a fibroblast cell to a stemcell-like cell having the capacity to divide unlimited times to formteratomas. The integration of a single copy of the lentiviral vector wassufficient to reprogram the fibroblast cell. In one embodiment, thelentiviral vector comprising the transcription factors is notintegrated.

The reprogramming of fibroblasts to an ESC-like state, pioneered byYamanaka and colleagues, has advanced stem cell research (Takahashi andYamanaka, 2006, Cell 126, 663-676) by circumventing certain obstacles.Takahashi and Yamanaka described retroviral transfer of fourtranscription factors (OCT4, SOX2, KLF4, and c-MYC) into fibroblasts,resulting in extensive reprogramming of the fibroblast epigenetic stateand transcriptome towards a state reminiscent of ES cells. Thefibroblasts employed in those studies were highly engineered, featuringan antibiotic resistance cassette knocked into the Fbx15 locus, allowingantibiotic selection of pluripotent clones that possessed broaddifferentiative repertoire in teratoma assays, including the ability todifferentiate into cells of all 3 germ layers. The cells created by thisbreakthrough have been termed induced pluripotent stem (iPS) cells todistinguish their method of derivation from that of ES cells. These socalled ‘induced Pluripotent Stem (iPS) cells’ derived from mouse(Maherali et al., 2007, Cell Stem Cell 1, 55-70; Okita et al., 2007,Nature 448, 313-317; Wernig et al., 2007, Nature 448, 318-324) or humanfibroblasts (Takahashi et al., 2007, Cell 131, 861-872; Yu et al., 2007,Science 318, 1917-1920) have demonstrated that an entire organism can bederived from readily accessible post-natal somatic cells. iPS cellsprovide a powerful in vitro model system for the study of the molecularmechanisms of reprogramming (Brambrink et al., 2008, Cell Stem Cell 2,151-159; Meissner et al., 2008, Nature; Mikkelsen et al., 2008, Nature454, 49-55; Mikkelsen et al., 2007, Nature 448, 553-560; Stadtfeld etal., 2008, Cell Stem Cell 2, 230-240) and have been successfullyemployed in proof-of-principle cell-based therapies in mouse models ofdisease (Hanna et al., 2007, Science 318, 1920-1923 Wernig et al., 2008,Proc Natl Acad Sci USA 105, 5856-5861).

However, to date the derivation of iPS cells has required multipleindividual viral vectors to deliver the constellation of transcriptionfactors (typically OCT4, SOX2, KLF4, and c-MYC) required to inducereprogramming. The application of sufficient quantities of each virusneeded to deliver four factors simultaneously to each target cellresults in high numbers of genomic integrations in successfullyreprogrammed progeny. This presence of multiple viral integrationsacross the genome prohibits their genetic elimination to produce saferiPS cells (Takahashi and Yamanaka, 2006, Cell 126, 663-676).Furthermore, the use of multiple vectors severely limits its use forhuman clinical applications.

The inventors presented herein the generation of a single lentiviralvirion (sometimes also known as capsid or vector) expressing thetranscription factors necessary to induced reprogramming a somatic cell,turning it to an induced programmed stem cell (iPS) cell. For example,by expressing OCT4, SOX2, KLF4, and c-MYC from a single multicistronicRNA transcript. The coding sequences for the four transcription factors,OCT4, SOX2, KLF4, and c-MYC, are constructed into a cassette termedherein as the ‘stem cell cassette’ wherein the coding sequences of OCT4,SOX2, KLF4, and c-MYC are ligated in one embodiment, in tandem and inthe sense orientation such that the coding sense strands of each geneare on the same strand in the cassette. An example of a recombinantlentiviral gene transfer plasmid comprising the stem cell cassette ispHAGE-STEMCCA. The lentiviral plasmid pHAGE is a third-generationself-inactivating lentiviral vector (A. B. Balazs and R. C. M.,unpublished work). The detailed DNA structures of two examples ofpHAGE-STEMCCA are described herein in FIGS. 6 and 7, and SEQ. ID. Nos.23 and 24. These pHAGE-STEMCCA plasmids are packaged into lentivirusesin 293T cells.

This stem cell cassette achieves expression of four individualtranscription factor proteins from a single multicistronic mRNAcontaining an IRES element separating two fusion cistrons. One fusioncistron comprises Oct4 and Klf4 coding sequences in tandem. The secondcistron comprises Sox2 and c-Myc coding sequences in tandem. Sequencesencoding ‘self-cleaving’ A peptides (FIG. 1A) separate the two genes ineach fusion cistron. The two fusion cistrons are joined so that the Oct4and Klf4 coding sequences are fused to Sox2 and c-Myc coding sequences,but separated by an IRES element. The single multi-cistronic mRNAcontains the mRNAs of the following proteins and peptides in this order:Oct4, ‘self-cleaving’ 2A peptide F2A, Klf4, Sox2, E2A and c-Myc (FIG.1A). The IRES element is found between the sequences of Sox2 and Klf4(FIG. 1A).

Furthermore, the inventors demonstrate efficient derivation oftransgene-free iPS cells using an excisable polycistronic lentiviralvector, pHAGE-STEMCCA-loxP. In other words, after the integration of thepolycistronic lentiviral vector that brought about the deprogramming ofdifferentiated cells, the integrated polycistronic lentiviral vector canbe efficiently excised to leave behind lentivirus-free iPS cells. Adirect comparison of iPS cell clones before and after excision revealsthat removal of the reprogramming vector markedly improves thedevelopmental potential of iPS cells and significantly augments theircapacity to undergo directed differentiation in vitro.

Furthermore, a specific marker gene is incorporated into thepolycistronic lentiviral vector for the purpose of monitoring thepresence or absence of the lentivirus in the integrate iPS cells orlentivirus-free iPS cells respectively. The specific marker gene can beone that expresses optically visible proteins such as the greenfluorescent protein or the “cherry” fluorescent protein described hereinor an enzyme whose activity can be assayed, e.g. thymidine kinase.

In one embodiment, the stem cell cassette comprises only four genesselected from the group consisting of Oct4, Klf4, Sox2, Lin28, Nanog andc-Myc. In another embodiment, only three genes selected from the groupconsisting of Oct4, Klf4, Sox2, Lin28, Nanog and c-Myc, e.g. Oct4, Klf4,and Sox2; Oct4, Klf4, and c-Myc; or Oct4, Sox2 and Lin28. In theseembodiments where only three genes are used, a specific marker genetakes the place of the fourth gene in the cassette.

The transcript elements in the recombinant lentiviral gene transferplasmid comprising the stem cell cassette can be expressedconstitutively or induced. For example, the inventors generated twoforms of pHAGE-STEMCCA in which the multi-cistronic transcript is drivenby either a constitutive EF1α promoter or a doxycycline (dox)-inducibleTetO-miniCMV promoter. Both vectors resulted in the expression of allfour individual proteins (OCT4, SOX2, KLF4, and c-MYC) as detected bywestern blot and immunohistochemistry (FIG. 1B and FIG. 1C).

The inventors tested the capacity of pHAGE-STEMCCA to derive iPS clonesfrom mouse embryonic or post-natal fibroblasts. Due to the large size ofthe proviral genome of these vectors (>9 Kb), pHAGE-STEMCCA viral titers(2-3×10⁸/ml) were lower than those obtained using mono-cistronic pHAGEvectors (5×10⁹/ml). Nevertheless, mouse embryonic fibroblasts (MEFs) andtail-tip fibroblasts (TTFs) transduced with the constitutive EF1αSTEMCCA construct showed a dramatic change in morphology already evident6 days post-infection and formed colonies that were clonally expandedand displayed the typical morphology of ES cell colonies (FIG. 2A).

For dox-inducible reprogramming, TTFs from a Sox2-GFP Rosa26-M2rtTAdouble knock-in mouse were transduced with pHAGE-STEMCCA that has adoxycycline (dox)-inducible TetO-miniCMV promoter. Cells from this mouseexpress rtTA constitutively and express GFP only upon activation of theSox2 locus, which is silent in fibroblasts but active in ES cells. TTFstransduced with pHAGE-Tet-STEMCCA were exposed to doxycycline andchanges in cell morphology were evident 6-8 days post induction withcolonies appearing at day 12-14 (FIG. 2B). iPS colonies derived usingeither the constitutive (EF1α-) or inducible (Tet-) pHAGE-STEMCCA vectorshowed similarly positive alkaline phosphatase (AP) and stage-specificembryonic antigen 1(SSEA1) staining as well as consistent and strong GFPexpression from the Sox2 locus (FIG. 2A and FIG. 2B). In addition, iPSclones generated with either vector expressed a variety of classic EScell marker genes. These genes are not expressed in fibroblasts prior toreprogramming (FIG. 2C). Furthermore each iPS clone evidenced thecorrect transmission of the full lentiviral vector genome and containedonly 1-3 integrated viral copies (FIG. 2D).

The inventors showed that the expression of this ‘stem cell cassette’transcript in mouse fibroblasts accomplishes the efficient derivation ofiPS cells with a single viral integration. Using the loxp/Cretechnology, the integrated stem-cell cassette and viral genome wereefficiently removed as shown in FIGS. 8C and 8D where the exogenous DNAwas not detected by qRT-PCR and Southern Blots. The singlenon-integrating polycistronic lentiviral viral vector provides advancesfor the potential application of iPS technology in human clinicaltrials.

Reprogramming mediated by an Oct4-Klf4-Sox2-c-Myc stem-cell cassettecontaining lentiviral vector such pHAGE-STEMCCA recombinant lentiviralgene transfer plasmid offers several advances over existing multi-vectorapproaches. By using a single vector-based approach, the possibility toinduce reprogramming with limited numbers of viral integrations can beachieved. One can obtain a uniform population of reprogrammed cells,i.e., one does not have to worry about only infecting a cell with 1, 2,or 3 genes encoding the transcription factors as opposed to all desiredtranscription factors, for example, Oct4-Klf4-Sox2-c-Myc, with a singleinfection. Indeed, the inventors were able to derive iPS clones withonly a single integrated viral copy (FIG. 2D). This is in markedcontrast to previous reports using multiple vectors, each with adifferent transcription factor, which required >15 viral integrations(Takahashi and Yamanaka, 2006, Cell 126, 663-676; Wernig et al., 2007,Nature 448, 318-324).

Accordingly, the present invention provides a vector system comprising:(a) a first vector containing a lentiviral gag gene encoding alentiviral Gag protein, wherein the lentiviral gag gene is operablylinked to a promoter and a polyadenylation sequence, (b) a second vectorcontaining an env gene encoding a functional envelope protein, whereinthe env gene is operably linked to a promoter and a polyadenylationsequence; (c) a lentiviral pol gene encoding a lentiviral Pol protein,wherein the pol protein is at least an integrase, and the pol gene is onthe first or second vectors or on at least a third vector, wherein thelentiviral pol gene is operably linked to a promoter and apolyadenylation sequence; wherein the at least first, second and thirdvectors do not contain sufficient nucleotides to encode the lentiviralGag and Pol and the envelope protein on a single vector; and wherein thevectors do not contain nucleotides of the lentiviral genome referred toas a packaging segment to effectively package lentiviral RNA; andwherein the lentiviral proteins and the envelope protein when expressedin combination form a lentivirus virion containing an envelope proteinaround a lentiviral capsid; and (d) a packaging gene transfer plasmidcomprising a stem cell cassette nucleic acid sequence encoding: a Oct4gene; a Klf4 gene; a Sox2 gene; a c-Myc gene; a first ‘self-cleaving’ 2Apeptide; a second ‘self-cleaving’ 2A peptide; an internal ribosome entrysite (IRES); wherein the nucleic acid sequence is operably linked to apromoter, wherein the sequences encoding Oct4, Klf4, Sox2, c-Myc, firstand second ‘self-cleaving’ 2A peptides, and the IRES are transcribedfrom the promoter as a multi-cistronic RNA.

In one embodiment, the packaging gene transfer plasmid comprising a stemcell cassette nucleic acid sequence encoding: a first gene; a secondgene; a third gene; an optional fourth gene; a first ‘self-cleaving’ 2Apeptide; a second ‘self-cleaving’ 2A peptide; and an internal ribosomeentry site (IRES); wherein the first, second, third and optional fourthgenes are selected from the group consisting of Oct4, Klf4, Sox2, c-Myc,Lin28, and Nanog; and wherein the first, second, third and optionalfourth genes are not identical; and wherein if the optional fourth geneis selected from the group consisting of Oct4, Klf4, Sox2, c-Myc, Lin28,and Nanog, a marker gene is included in its place, wherein the markergene encodes an optically visible protein or an enzyme.

In one embodiment, the packaging gene transfer plasmid comprises a stemcell cassette nucleic acid sequence encoding four transcription factorgenes selected from a group consisting of Oct4, Klf4, Sox2, c-Myc, Lin28and Nanog; a first ‘self-cleaving’ 2A peptide; a second ‘self-cleaving’2A peptide; an internal ribosome entry site (IRES); wherein the nucleicacid sequence is operably linked to a promoter, wherein the sequencesencoding the four transcription factor genes, first and second‘self-cleaving’ 2A peptides, and the IRES are transcribed from thepromoter as a multi-cistronic RNA.

In one embodiment, the packaging gene transfer plasmid comprises a stemcell cassette nucleic acid sequence encoding three transcription factorgenes selected from a group consisting of Oct4, Klf4, Sox2, c-Myc, Lin28and Nanog; a first ‘self-cleaving’ 2A peptide; a second ‘self-cleaving’2A peptide; an internal ribosome entry site (IRES); wherein the nucleicacid sequence is operably linked to a promoter, wherein the sequencesencoding the three transcription factor genes, first and second‘self-cleaving’ 2A peptides, and the IRES are transcribed from thepromoter as a multi-cistronic RNA.

In one embodiment, the packaging gene transfer plasmid comprises a stemcell cassette nucleic acid sequence encoding: three transcription factorgenes selected from a group consisting of Oct4, Klf4, Sox2, c-Myc, Lin28and Nanog; a marker gene, a first ‘self-cleaving’ 2A peptide; a second‘self-cleaving’ 2A peptide; an internal ribosome entry site (IRES);wherein the nucleic acid sequence is operably linked to a promoter,wherein the sequences encoding the three transcription factor genes, themarker gene, first and second ‘self-cleaving’ 2A peptides, and the IRESare transcribed from the promoter as a multi-cistronic RNA.

In one embodiment, the packaging gene transfer plasmid further comprisestwo Cre-LoxP sequences that flank the stem cell cassette nucleic acidsequence (see schematic design in FIG. 8A).

In one embodiment, the packaging gene transfer plasmid further comprisesa Cre-ERT2 sequence. The Cre-ERT2 encodes a Cre recombinase (Cre) fusedto a mutant estrogen ligand-binding domain (ERT2) that requires thepresence of tamoxifen for activity. Excision of the integrated viralvector equipped with Cre-LoxP sites can be induced with theadministration of tamoxifen.

In one embodiment, the integrase of the vector system has been modifiedso that it is not capable of integration.

In one embodiment, the vector system described herein compriseslentivirus selected from the group consisting of HIV, HIV-2, FIV, andSIV.

In one embodiment, the vector system described herein compriseslentivirus wherein the env gene encodes an envelope from a differentvirus, and is of a different source from the gag and pol genes.

In another embodiment, the present invention also provides a lentiviralvector particle capable for reprogramming a somatic cell to astem-cell-like cell, the vector particle comprises a nucleic acidsequence comprising a sequence encoding: (a) a Oct4 gene; (b) a Klf4gene; (c) a Sox2 gene; (d) a c-Myc gene; (e) a first ‘self-cleaving’ 2Apeptide; (f) a second ‘self-cleaving’ 2A peptide; (g) an internalribosome entry site (IRES); wherein the nucleic acid sequence isoperably linked to a promoter, and wherein the sequences encoding theOct4, Klf4, Sox2, c-Myc, first and second ‘self-cleaving’ 2A peptides,and the IRES are transcribed from the promoter as a multi-cistronic RNA.

In some embodiments, the nucleic acid sequence encodes a Nanog and/orLin 28 gene.

In some embodiments, the nucleic acid sequence encodes several types oftranscription factors sufficient to reprogram a somatic cell into aninduced pluripotent stem cell that have the characteristics of a stemcell, for example having self renewal capability and/or expressesembryonic stem cell markers that are well known in the art and alsodescribed herein. The several types of transcription factors can beselected from the group consisting of OCT4, KLF4, SOX2, c-MYC, NANOG andLIN28. In some embodiments, at least three types of transcriptionfactors are selected. Various combinations of transcription factors ofOCT4, KLF4, SOX2, c-MYC, NANOG AND LIN 28 are contemplated. For example,OCT4, KLF4, SOX2, and c-MYC are selected and encoded in the nucleic acidsequence described herein.

In one embodiment, the present invention provides a lentiviral vectorparticle capable for reprogramming a somatic cell to a lessdifferentiated state. This can range from a pluripotent stage to arelatively more differentiated stage. The key is that the cell is lessdifferentiated than the original cell. In this manner one can reprogramcells to a desired state. The vector particle comprising a nucleic acidsequence comprising a sequence encoding: (a) a first gene; (b) a secondgene; (c) a third gene; (d) an optional fourth gene; (e) a first‘self-cleaving’ 2A peptide; (f) a second ‘self-cleaving’ 2A peptide; and(g) an internal ribosome entry site (IRES); wherein the first, second,third and optional fourth genes can be selected from the groupconsisting of Oct4, Klf4, Sox2, c-Myc, Lin28, and Nanog; wherein thefirst, second, third and optional fourth genes are not identical;wherein the nucleic acid sequence is operably linked to a promoter,wherein the sequences encoding the first, second, third and optionalfourth genes, first and second ‘self-cleaving’ 2A peptides, and the IRESare transcribed from the promoter as a multi-cistronic RNA. Othercombinations of reprogramming genes are knows and the present vectorsystem can be used with any of them.

In some embodiments, if the fourth optional gene is not selected, amarker gene is included in its place, wherein the marker gene encodes anoptically visible protein or an enzyme.

In some embodiments, alternate slice variants, functional conservativeamino acid substitutions and truncations of these transcription factorsare also contemplated (Atlasi Y., Stem Cells. 2008 Epub. September 11;A. E. F. Smith and K. G. Ford, Nucleic Acids Res. 2005, 33:6011-23; T.K. Nowling, J Biol Chem, 275: 3810-3818; Kit-Ling Sze, J. CellularPhysiology, 214:334-344). In some embodiments, family members of thesetranscription factors are used. For example, OCT4 is of the POU familyof transcription factors. POU proteins are eukaryotic transcriptionfactors containing a bipartite DNA binding domain referred to as the POUdomain. The various members of the POU family have a wide variety offunctions, all of which are related to the development of an organism.POU proteins are: POU1F1, POU2F1, POU2F2, POU2F3, POU3F1, POU3F2,POU3F3, POU3F4, POU4F1, POU4F2, POU4F3, POU5F1, POU6F1, and POU6F2.

In some embodiments, these transcription factor genes are derived fromhuman. In other embodiments, these transcription factor genes arederived from other mammals such as mouse, rat, and also other organismsuch as the nematode worm Caenorhabditis elegans.

In one embodiment, the somatic cell is a mammalian cell. In oneembodiment, the somatic cell is a mammalian cell derived from internalorgans-heart, kidney, liver, lungs, bladder, intestines; skin, bones,blood, cartilage and connective tissues.

In one embodiment, the sequences encoding the four genes, e.g. Oct4,Klf4, Sox2, and c-Myc, in the lentiviral vector particle are arranged intandem, wherein the genes are oriented in the sense direction, andwherein the genes are arranged in any order. This form astem-cell-cassette. Some examples of tandem arrangement areOct4-Klf4-Sox2-c-Myc, Oct4-Sox2-Klf4-c-Myc, Sox2-Klf4-Oct4-c-Myc,Klf4-Sox2-Oct4-c-Myc, and c-Myc-Sox2-Oct4-Klf4. Similarly, the sequencesencoding the three transcription factor genes and a marker gene in thelentiviral vector particle are arranged in tandem, wherein the genes areoriented in the sense direction, and wherein the genes are arranged inany order. In all embodiments, the end of the 5′ upstream (front) geneis fused to the beginning of the immediate downstream gene, thus thegenes are oriented in the sense direction. Following transcription, amulti-cistronic mRNA can be translated to result in four individualproteins.

SOX2 is the human SRY (sex determining region Y)-box 2, (GenbankAccession No. BC013923; NM_(—)003106.2; cDNA clone MGC:2413IMAGE:2823424, SEQ. ID. No. 3). It is also known as ANOP3, MCOPS3, andMGC2413. This intronless gene encodes a member of the SRY-relatedHMG-box (SOX) family of transcription factors involved in the regulationof embryonic development and in the determination of cell fate. Theproduct of this gene is required for stem-cell maintenance in thecentral nervous system, and also regulates gene expression in thestomach.

c-MYC is the human v-myc myelocytomatosis viral oncogene homolog(avian). It is also known as MYC and bHLHe39 (Genbank Accession No.NM_(—)002467.3, SEQ. ID. No. 4). The protein is a multifunctional,nuclear phosphoprotein that plays a role in cell cycle progression,apoptosis and cellular transformation. It functions as a transcriptionfactor that regulates transcription of specific target genes. Mutations,overexpression, rearrangement and translocation of this gene have beenassociated with a variety of hematopoietic tumors, leukemias andlymphomas, including Burkitt lymphoma.

KLF4 is the human Kruppel-like factor 4 (gut) (Genbank Accession No.NM_(—)004235.4, SEQ. ID. No. 5). It is also known as EZF and GKLF. It isexpressed transiently in certain mesenchymal cell and is an inhibitor ofcell growth.

OCT4 is POU class 5 homeobox 1 (Genbank Accession No. NM_(—)002701.4,SEQ. ID. No. 6; NM_(—)203289.3, SEQ. ID. No. 7). It is also known asOCT3, OTF3, OTF4, POU5F1, and MGC22487.

NANOG is a transcription factor critically involved with self-renewal ofundifferentiated embryonic stem cells. NANOG is a gene expressed inembryonic stem cells (ESCs) and is thought to be a key factor inmaintaining pluripotency. NANOG is thought to function in concert withother factors such as POU5F1 and SOX2 to establish ESC identity.(Genbank Accession No. NM_(—)024865, SEQ. ID. No. 47).

LIN28 is the human homolog of lin-28 of worms. It is also known asCSDD1; LIN-28; LIN28A; ZCCHC1; FLJ12457. (Genbank Accession No.NM_(—)024674.4, SEQ. ID. No. 48).

In one embodiment, the sequence encoding an internal ribosome entry site(IRES) is between the second and third genes in the tandem arrangementof the four genes, e.g. Oct4, Klf4, Sox2, and c-Myc. However, othercombinations are known in the art and are envisioned herein.

An internal ribosome entry site, abbreviated IRES, is a nucleotidesequence that allows for translation initiation in the middle of amessenger RNA (mRNA) sequence as part of the greater process of proteinsynthesis. Usually, in eukaryotes, translation can only be initiated atthe 5′ end of the mRNA molecule, since 5′ cap recognition is requiredfor the assembly of the initiation complex. IRES as cis-acting RNAsequences are able to mediate internal entry of the 40S ribosomalsubunit on some eukaryotic and viral messenger RNAs upstream of atranslation initiation codon. These sequences are very diverse and arepresent in a growing list of mRNAs. The IRES database is a comprehensiveWorld Wide Web resource for internal ribosome entry sites and presentscurrently available general information as well as detailed data foreach IRES. It is a searchable, periodically updated collection of IRESRNA sequences. Sequences are presented in FASTA form and hotlinked toNCBI GenBank files. Several subsets of data are classified according tothe viral taxon (for viral IRESes), to the gene product function (forcellular IRESes), to the possible cellular regulation or to thetrans-acting factor that mediates IRES function. This database isaccessible at at the World Wide Web site of “ifr31w3” “period”“Toulouse” “period” “inserm” “period” “fr” “/IRESdatabase/” and at theWorld Wide Web site of “rangueil” “period” “inserm” “period” “fr”“/IRESdatabase/”.

Use of the IRES sequences are well known in the art. For example, inU.S. Pat. Nos. 4,937,190, 6,159,709, and 6,171,821. One skilled in theart would be able to search for known IRES sequences and incorporate anIRES element between the coding sequences of two genes. For example, theIRES sequence of encephalomyocarditis virus can be isolated from theLXIN retroviral vector, pLXIN vector, (Clontech, Palo Alto, Calif.) withrestriction enzyme digestions, isolated and then inserted into thestem-cell-cassette described. Alternatively, commercial lentiviralvectors with IRES can be used. The coding sequences of the four genes:Oct4, Klf4, Sox2, and c-Myc can be inserted into these commerciallentiviral vectors. Examples of such commercial lentiviral vectorsinclude but not limited to pReceiver-Lv31, -Lv32, -Lv33, -Lv35, -Lv36,-Lv40, -Lv43, -Lv44, and -Lv47 from Capital Biosciences, Inc.,pLenti4/V5-DEST™, pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ(Invitrogen), and TREAutoR2 lentiviral vectors described in D. Markusicet. al. (Nucleic Acids Research 2005 33(6):e63).

In one embodiment, the sequence encoding the first ‘self-cleaving’ 2Apeptide is between the first and second genes in the tandem arrangementof the four genes, e.g. Oct4, Klf4, Sox2, and c-Myc. In anotherembodiment, the sequence encoding the second ‘self-cleaving’ 2A peptideis between the third and fourth genes in the tandem arrangement the fourgenes, e.g. Oct4, Klf4, Sox2, and c-Myc.

The use of the 2A peptide in multi-cistronic constructs has emerged asan attractive alternative to the IRES. Like the IRES, the 2A peptide wasidentified among picornaviruses but in a different sub-group, theAphthoviruses, a typical example of which is the Foot-and-mouth diseasevirus (Robertson B H, et. al., J Virol 1985, 54:651-660) 2A-likesequences have since been found in other Picornaviridae like the Equinerhinitis A virus, as well as unrelated viruses such as the Porcineteschovirus-1 and the insect Thosea asigna virus (TaV) (Donnelly M L,et. al., J. Gen. Virol. 2001, 82:1027-1041). In such viruses, multipleproteins are derived from a large polyprotein encoded by a single openreading frame. The 2A peptide mediates the co-translational cleavage ofthis polyprotein at a single site that forms the junction between thevirus capsid and replication polyprotein domains.

The 2A sequences are relatively short peptides (of the order of 20 aminoacids long, depending on the virus of origin) containing the consensusmotif D-V/I-E-X-N-P-G-P (SEQ ID NO: 52; preferred embodiments disclosedas SEQ ID NOS 1-2). They were originally thought to mediate theautocatalytic proteolysis of the large polyprotein, but are nowunderstood to act co-translationally, by preventing the formation of anormal peptide bond between the glycine and last proline, resulting inthe ribosome skipping to the next codon (Donnelly M L, et. al., J GenVirol 2001, 82:1013-1025) and the nascent peptide cleaving between theGly and Pro. After cleavage, the short 2A peptide remains fused to theC-terminus of the ‘upstream’ protein, while the proline is added to theN-terminus of the ‘downstream’ protein. Based on highly inefficientpeptide bond formation between Gly and Pro residues within the 2Apeptide, placement of 2A peptide sequence as a linker region betweentandem cDNA's, e.g. between Oct4 and Sox2, allows the stoichiometrictranslation of multiple unfused protein products.

Examples of self-processing peptides 2A peptides include but not limitedto: F2A: VKQTLNNFDLLKLAGDVESNPGP (SEQ. ID. No. 8), E2A:QCTNYALLKLAGDVESNPGP (SEQ. ID. No. 9), T2A: EGRSLLTCGDVEENPGP (SEQ. ID.No. 10), and P2A: ATNFSLLKQAGDVEENPGP (SEQ. ID. No. 11)

In one embodiment, the coding sequence for a 2A peptide is 5′ TGG GCCAGG ATT CTC CTC GAC GTC ACC GCA TGT TAG CAG ACT TCC TCT GCC CTC TCC ACTGCC3′ (SEQ. ID. No. 12).

In one embodiment, the first ‘self-cleaving’ 2A peptide between thefirst and second genes in the stem cell cassette is selected from thegroup consisting of F2A, E2A, T2A and P2A. In another embodiment, thesecond ‘self-cleaving’ 2A peptide between the third and fourth genes inthe stem cell cassette is selected from the group consisting of F2A,E2A, T2A and P2A. In a further embodiment, the second ‘self-cleaving’ 2Apeptide is different from the first ‘self-cleaving’ 2A peptide.

In one embodiment, the promoter for the described stem cell cassette isinducible. In one embodiment, the promoter is tetracycline regulated. Inanother embodiment, the promoter is a TetO/miniCMV promoter. TheTetO/miniCMV promoter comprises the tetracycline-responsivetranscriptional regulatory element of Escherichia coli (tetO) sequencelinked to a minimal CMV promoter (miniCMV). The minimal CMV promoter isthe core immediate early promoter of the human cytomegalovirus, in whichthe enhancer sites have been deleted. The TetO is bound by the Tetrepressor protein (tetR) and gene transcription from the miniCMVpromoter is blocked. The repression in relieved with tetracycline or itsanalogue such as doxycyclin. In the presence of tetracycline, thetetracylin repressor binds tetracycline, which binding displaces therepressor from the tetracycline operator sequence, so repression isrelieved and transcription can begin. Construction of a TetO/miniCMVpromoter vector is well known in the art, for example, in Bohl et al.(1998) Blood 92 (5): 1512-1517 and Haberman et al. (1998) Gene Ther. 5:1604-1611. The tetracycline operator sequence is described by Baron U.et al., Nucleic acid research, Vol. 17, p. 3605-3606 (1995). The minimalCMV promoter construction and use are described in U.S. Pat. No.6,368,825 and it is hereby incorporated by reference in its entirety.

In other embodiments, the promoter comprises hormone-responsive elements(HREs), metal-responsive elements (MREs), heat shock-responsive elements(HSREs), cytokine responsive elements or interferon-responsive elements(IREs) operably linked to a promoter, e.g. miniCMV. Expressions of genesoperably linked to such promoters are induced in the presence ofhormone, heavy metal, increases in temperature or interferonrespectively. For example, a glucocorticoid-responsive element (GRE) isrecognized and bound by the glucocorticoid/receptor complex and geneexpression is induced. In some embodiments, the responsive elementscomprise two or more responsive elements (see U.S. Pat. No. 5,877,018).Regulation of gene expression by hormone, heavy metal, temperature orinterferon is well known in the art. One skilled in the art canincorporate the responsive element of choice into the construct of alentiviral vector described herein. For example, the GRE consensussequence is 5′-GATCTGGTACAGGATGTTCTAGCTACG-3′ (SEQ. ID. No: 13) or MREconsensus sequence is 5′-GATCTTGCGCCCGGCCCG-3′ (SEQ. ID. No: 14). can beOther examples of inducible promoter are described in U.S. PatentApplication 20050227285 and U.S. Pat. No. 5,877,018, and thesereferences are hereby incorporated by reference in their entirety.

In one embodiment, the promoter for the described stem cell cassette isconstitutive. In one embodiment, the promoter is EF-1alpha. In anotherembodiment, the promoter is beta-actin.

One of ordinary skill in the art can construct the stem cell cassette ina lentiviral vector. Conventional polymerase chain reaction (PCR)cloning techniques can be used to generate the isolated DNA sequenceencoding three or four transcription factors, e.g. Oct4, Klf4, Sox2, andc-Myc. Ideally each PCR primer should have at least 15 nucleotidesoverlapping with its corresponding templates at the region to beamplified. The polymerase used in the PCR amplification should have highfidelity such as Strategene's PfuUltra™ polymerase for reducing sequencemistakes during the PCR amplification process. For ease of ligating thePCR amplified coding sequence to the leniviral vector, the PCR primersshould also have distinct and unique restriction digestion sites ontheir flanking ends that do not anneal to the DNA template during PCRamplification. The choice of the restriction digestion sites for eachpair of specific primers should be such that the coding nucleic acidsare is in-frame and will encode the proteins OCT4, KLF4, SOX2, and c-MYCrespectively from beginning to end with no stop codons. At the same timethe chosen restriction digestion sites should not be found within theSEQ. ID. Nos.: 3-7, and SEQ. ID. Nos.: 47-48.

In one embodiment, the primers Oct4 5′ NotI5′CACCGGCGGCCGCCATGGATCCTCGAACCTGGCTAAGCTTCCAAG-3′ SEQ. ID. No. 15 andOct4-F2A 3′ 5′CTTGAGAAGGTCAAAATTCAAAGTCTGTTTCACGCCACTTCCGTTTGAATGCATGGGAGAGCCCAGAGCAG-3′ SEQ. ID. No. 16 are used to PCR clone thecDNA of OCT4 from template SEQ. ID. No. 6 or SEQ. ID. No. 7. The primerOct4 5′ NotI has the restriction NotI site incorporated at its 5′ end.The primer Oct4-F2A 3′ has the coding sequence of F2A peptide at the 3′end. The PCR product has the restriction NotI site at the 5′ endfollowed by the Oct4 and ending with the coding sequence of F2A peptide.

In one embodiment, the primers F2A-Klf45′5′AAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCCATGGCTAGCGACGCTCTGCTCCC-3′ SEQ. ID. No. 17 and Klf4 3′ BamHI5′TTTGGATCCTTAAAAGTGCCTCTTCATGTGTAAGGCAAG-3′ SEQ. ID. No. 18 are used toPCR clone the cDNA of Klf4 from template SEQ. ID. No. 5. The primer Klf43′ BamHI has the restriction BamHI site incorporated at its 3′ end. Theprimer F2A-Klf4 5′ has the coding sequence of F2A peptide at the 5′ end.The PCR product has the coding sequence of F2A peptide at the 5′ endfollowed by the Klf4 and ending with the restriction BamHI site at the3′ end.

In one embodiment, the fusion cistron comprising OCT4, FA2 and KLF4arranged in tandem and oriented in the sense direction is constructed byPCR amplification of a mixture of the purified PCR products of the OCT4PCR cloning reaction and the PCR products of the KLF4 PCR cloningreaction at a ratio of 1:1. The primers Oct4 5′ NotI and Klf4 3′ BamHIare used to PCR clone the chimeric DNA sequence comprising Oct4 at the5′ end followed by FA2 peptide and ending with Klf4. This PCR product isflanked by the restriction NotI site at the 5′ and the restriction BamHIsite at the 3′ end. In one embodiment, this PCR product is digested withNotI and BamHI, and ligated into a previously NotI/BamHI digestedlentiviral vector, e.g. pHAGE2, wherein the ligation is upstream of theIRES of the lentiviral vector.

In one embodiment, the primers Sox2 5′ NdeI 5′GGTTTCTTACATATGATGTATAACATGATGGAGACGGAGCTGAAG-3′ SEQ. ID. No. 19 andSox2-E2A 3′ TTTCAACATCGCCAGCGAGTTTCAACAAAGCGTAGTTAGTACATTGCCCACTACCCATGTGCGACAGGGGCAGTGTGCCGTTAATGGCCG-3′ SEQ. ID. No. 20 are used to PCR clonethe cDNA of Sox2 from template SEQ. ID. No. 3. The primer Sox2 5′ NdeIhas the restriction NdeI site incorporated at its 5′ end. The primerSox2-E2A 3′ has the coding sequence of E2A peptide at the 3′ end. ThePCR product has the restriction NdeI site at the 5′ end followed by theSox2 and ending with the coding sequence of E2A peptide.

In one embodiment, the primers E2A-cMyc 5′5′-CTTTGTTGAAACTCGCTGGCGATGTTGAAAGTAACCCCGGTCCTATGCCCCTCAACGTGAACTTCACCAACAGGAACTATG-3′ SEQ. ID. No. 21 and cMyc 3′ ClaI5GGTTTATCGATTTATGCACCAGAGTTTCGAAGCTGTTC-3′ SEQ. ID. No. 22 are used toPCR clone the cDNA of c-Myc from template SEQ. ID. No. 4. The primerE2A-cMyc 5′ has the coding sequence of E2A peptide incorporated at its5′ end. The primer cMyc 3′ ClaI has the restriction ClaI site at the 3′end. The PCR product has the coding sequence of E2A peptide at the 5′end followed by the c-Myc and ending with the restriction ClaI site.

In one embodiment, the fusion cistron comprising Sox2, EA2 and c-Mycthat are arranged in tandem and oriented in the sense direction isconstructed by PCR amplification of a mixture of the purified PCRproducts of the Sox2 PCR cloning reaction and the PCR products of thec-Myc PCR cloning reaction at a ratio of 1:1. The primers Sox2 5′ NdeIand cMyc 3′ ClaI are used to PCR clone the chimeric DNA sequencecomprising Sox2 at the 5′ end followed by EA2 peptide and ending withc-Myc. This PCR product is flanked by the restriction NdeI site at the5′ and the restriction ClaI site at the 3′ end. In one embodiment, thisPCR product is digested with NdeI and ClaI, and ligated into apreviously NdeI/ClaI digested lentiviral vector, e.g. pHAGE2, whereinthe ligation is downstream of IRES of the lentiviral vector.

In one embodiment, the primers E2A-mCherry 5′(5′-CTTTGTTGAAACTCGCTGGCGATGTTGAAAGTAACCCCGGTCCTATGGTGAGCAAGGGCGAGGAGGATAACATGGCC-3′ SEQ. ID. No. 41) and mCherry 3′ClaI(5′-ATCGATTTACTTGTACAGCTCGTCCATGCCGCCGGTG-3′ SEQ. ID. No. 42) are usedto PCR clone the cDNA of the mCherry gene from the template GENBANKAccession No. AY678264 (SEQ. ID. No. 49) which codes for a monomeric redfluorescent protein gene. This gene is an engineered variant ofmonomeric red fluorescent protein mRFP1 in GENBANK Accession No.AF506027 (Shaner, N. C., et al., 2004, Nat. Biotechnol. 22:1567-1572).The primer E2A-mCherry 5′ has the coding sequence of E2A peptideincorporated at its 5′ end. The primer mCherry 3′ ClaI has therestriction ClaI site at the 3′ end. The PCR product has the codingsequence of E2A peptide at the 5′ end followed by the mCHerry and endingwith the restriction ClaI site.

In one embodiment, the nucleic acid sequence of the lentiviral genetransfer plasmid is pHAGE-Tet-STEMCCA (SEQ. ID. No. 23). This lentiviralgene transfer plasmid is also knows aspHAGE2-TetOminiCV-Oct4F2aKlf4-IRES-Sox2E2AcMyc-W.

In one embodiment, the nucleic acid sequence of the lentiviral genetransfer plasmid is pHAGE-EF1α-STEMCCA vector (SEQ. ID. No. 24). Thislentiviral gene transfer plasmid is also knows aspHAGE2-EF1αFull-Oct4F2aKlf4-IRES-Sox2E2AcMyc-W.

In one embodiment, the nucleic acid sequence of the lentiviral genetransfer plasmid is pHAGE-EF1α-STEMCCA-LoxP-RedLight (SEQ. ID. No. 50).This lentiviral gene transfer plasmid is also knows aspHAGE-EF1α-STEMCCA-LoxP-RedLight orpHAGE2-EF1αFull-Oct4F2aKlf4-IRES-Sox2E2AmCherry-W-LoxP. This vector hasthe LoxP flanking the STEMCCA and the fourth gene in the cassette,c-Myc, has been replaced with a marker gene, mCherry, which codes for ared fluorescent protein.

Lentiviral vectors are a type of retrovirus that can infect bothdividing and nondividing cells because their preintegration complex(virus “shell”) can get through the intact membrane of the nucleus ofthe target cell. Lentiviruses can be used to provide highly effectivegene therapy as lentiviruses can change the expression of their targetcell's gene for up to six months. They can be used for nondividing orterminally differentiated cells such as neurons, macrophages,hematopoietic stem cells, retinal photoreceptors, and muscle and livercells, cell types for which previous gene therapy methods could not beused. Examples of lentiviruses are human immunodeficiency virus (HIV)(strain 1 and strain 2), simian immunodeficiency virus (SIV), felineimmunodeficiency virus (FIV), BLV, EIAV, CEV and visna virus. Of these,HIV and SIV are presently best understood. HIV is a very effectivelentiviral vector. A vector containing such a lentivirus core (e.g. gaggene) can transduce both dividing and non dividing cells.

Recently, attention has focused on lentiviral vectors such as thosebased upon the primate lentiviruses, e.g., human immunodeficiencyviruses (HIV) and simian immunodeficiency virus (SIV). HIV vectors caninfect quiescent cells in addition to dividing cells. Moreover, by usinga pseudotyped vector (i.e., one where an envelope protein from adifferent species is used), problems encountered with infecting a widerange of cell types can be overcome by selecting a particular envelopeprotein based upon the cell you want to infect. Moreover, in view of thecomplex gene splicing patterns seen in a lentiviruses such as HIV,multivalent vectors (i.e., those expressing multiple genes) having alentiviral core, such as an HIV core, are expected to be more efficient.Despite the advantages that HIV based vectors offer, there is still aconcern with the use of HIV vectors in view of the severity of HIVinfection. Thus, means for providing additional attenuated forms thatare less likely to revert to a wild type virus are desirable.

Variations can be made where multiple modifications are made, such asdeleting nef, rev, vif and vpr genes. One can also have the 3′ and 5′ U3deleted LTRs.

Lentiviruses are the only type of virus that is diploid; they have twostrands of RNA. For example, HIV contains a diploid single strandedpositive sense RNA-genome that is approximately 10 kb long. The ends areflanked with long terminal repeats (LTRs). A Psi-sequence is found nearthe 5′ end of the RNA-genome which is necessary for packaging viral RNAinto virus capsids to continue the infection of HIV in its host.However, the HIV's genetic information is integrated into the DNA of thehost cell, so its RNA must be converted into DNA inside of the host forviral replication to be successful. This is done by reversetranscription of the RNA into DNA, and some of the proteins that areessential for this process. Reverse transcriptase synthesizes the firststrand of DNA from the RNA template, and the host DNA polymerasesynthesizes the second strand to produce dsDNA. Thus, quiescent cells donot have the ability to perform this second step in the reversetranscription process, so the RNA is not turned into DNA in cells in theGO state. This is the reason for the limitation on gene therapy with HIVvectors. The DNA copy just made, which contains the genes gag, env, andpol, is inserted by integrase into the host genome. LTRs are alsonecessary for integration of the dsDNA into the host chromosome. LTRsalso serve as part of the promoter for transcription of the viral genes.Thus, the virus is protected from attack by the immune system. It isthis ability of the HIV to integrate its genetic material into a hostcell which scientists would like to harness to put towards gene therapy.It has been shown that the HIV vector has an even higher rate ofexpression in its hosts cells than other retroviruses. HIV gene therapyvectors also do not trigger immune reactions, making them veryattractive delivery systems.

The preintegration complex of the human immunodeficient virus (HIV),which allows the vector assess inside human cells, dividing ornon-diving, is composed of the enzyme integrase, the product of the vprgene (an accessory gene), and a protein encoded by the gag gene (anessential structural gene) called matrix. This matrix protein contains alocalization sequence which is recognized by the import machinery of thenucleus of a cell. The virus is surrounded by a lipid bilayer withprotruding membrane proteins. One of these proteins, gp120, isrecognized by the host helper T cell CD4 receptor protein. Then HIVbinds to a secondary receptor (CCR5 or CXCR4) and triggers a membranefusion-mechanism with the gp41 transmembrane protein. This allows thevirus assess to the cell interior and the virus content is released intothe cytoplasm of the cell. Once inside of the cell in the cytoplasm, thematrix protein of the HIV contains a localization sequence that isrecognized by the nuclear import machinery, which docks the complex at anuclear membrane pore. This enables the preintegration complex of theHIV lentiviral vector to pass into the nucleus.

The lentiviral virion (particle) is expressed by a vector systemencoding the necessary viral proteins to produce a virion (viralparticle). Preferably, there is at least one vector containing a nucleicacid sequence encoding the lentiviral Pol proteins necessary for reversetranscription and integration, operably linked to a promoter.Preferably, the Pol proteins are expressed by multiple vectors. There isalso a vector containing a nucleic acid sequence encoding the lentiviralGag proteins necessary for forming a viral capsid operably linked to apromoter. In one embodiment, the gag-pol genes are on the same vector.Preferably, the gag nucleic acid sequence is on a separate vector thanat least some of the pol nucleic acid sequence, still more preferably itis on a separate vector from all the pol nucleic acid sequences thatencode Pol proteins.

In one embodiment, the gag sequence does not express a functional MAprotein, i.e. the vector can still transduce cells in the absence of theentire MA or a portion thereof, if a myristylation anchor is provided.This can be accomplished by inactivating the “gene” encoding the MA byadditions, substitutions or deletions of the MA coding region.Preferably, this is done by deletion. Preferably, at least 25% of the MAcoding region is deleted, more preferably, at least 50% is deleted,still more preferably, at least 60%, even more preferably at least 75%,still more preferably, at least 90%, yet more preferably at least 95%and most preferably the entire coding region is deleted. However, inthat embodiment, a myristylation anchor (sequence) is still required.Preferably, the myristylation sequence is a heterologous (i.e.,non-lentiviral) sequence.

In another embodiment the lentiviral vector is another form ofself-inactivating (SIN) vector as a result of a deletion in the 3′ longterminal repeat region (LTR). Preferably, the vector contains a deletionwithin the viral promoter. The LTR of lentiviruses such as the HIV LTRcontains a viral promoter. Although this promoter is relativelyinefficient, when transactivated by e.g. tat, the promoter is efficientbecause tat-mediated transactivation increases the rate of transcriptionabout 100 fold. However, the presence of the viral promoter caninterfere with heterologous promoters operably linked to a transgene. Tominimize such interference and better regulate the expression oftransgenes, the lentiviral promoter is preferably deleted.

Preferably, the vector contains a deletion within the viral promoter.The viral promoter is in the U3 region of the 3′ LTR. A preferreddeletion is one that is 120 base pairs between ScaI and PvuI sites, e.g.corresponding to nucleotides 9398-9518 of HIV-1 proviral clone HXB2,encompassing the essential core elements of the HIV-1 LTR promoter (TATAbox, SP1 and NF-PB binding sites). After reverse transcription, thedeletion is transferred to the 5′ LTR, yielding a vector/provirus thatis incapable of synthesizing vector transcripts from the 5′ LTR in thenext round of replication. Thus, the vector of the present inventioncontains no mechanism by which the virus can replicate as it cannotexpress the viral proteins.

In another embodiment the vector is a tat deleted vector. This can beaccomplished by inactivating at least the first exon of tat by knowntechniques such as deleting it. Alternatively, one can extend the U3 LTRdeletion into the R region to remove the TAR element.

Variations can be made where the lentiviral vector has multiplemodifications as compared to a wildtype lentivirus. For example, withHIV being nef-, rev-, vpu-, vif- and vpr-. In addition one can haveMA-gag, 3′ and 5′ U3 deleted LTR and variations thereof.

The vector(s) do not contain nucleotides from the lentiviral genome thatpackage lentiviral RNA, referred to as the lentiviral packagingsequence. In HIV this region corresponds to the region between the 5′major splice donor and the gag gene initiation codon (nucleotides301-319).

The env, gag and pol vector(s) forming the particle preferably do notcontain a nucleic acid sequence from the lentiviral genome thatexpresses an envelope protein. Preferably, a separate vector contains anucleic acid sequence encoding an envelope protein operably linked to apromoter is used. This env vector also does not contain a lentiviralpackaging sequence. In one embodiment the env nucleic acid sequenceencodes a lentiviral envelope protein.

In another embodiment the envelope protein is not from the lentivirus,but from a different virus. The resultant particle is referred to as apseudotyped particle. By appropriate selection of envelopes one can“infect” virtually any cell. Thus, the vector can readily be targeted toa specific cell. For example, one can use an env gene that encodes anenvelope protein that targets an endocytic compartment such as that ofthe influenza virus, VSV-G, alpha viruses (Semliki forest virus, Sindbisvirus), arenaviruses (lymphocytic choriomeningitis virus), flaviviruses(tick-borne encephalitis virus, Dengue virus), rhabdoviruses (vesicularstomatitis virus, rabies virus), and orthomyxoviruses (influenza virus).

The preferred lentivirus is a primate lentivirus (U.S. Pat. No.5,665,577) or a feline immunodeficiency virus (FIV) (Poeschla, E. M., etal., 1998, Nat. Medicine 4:354-357). The pol/gag nucleic acid segment(s)and the env nucleic acid segment will when expressed produce an emptylentiviral particle. By making the above-described modifications such asdeleting the tat coding region, the MA coding region, or the U3 regionof the LTR, the possibility of a reversion to a wild type virus has beenreduced.

A desired family of heterologous nucleic acid segments (sometimesreferred to as the target molecule) can be inserted into the emptylentiviral particles by use of a plurality of vectors each containing anucleic acid segment of interest and a lentiviral packaging sequencenecessary to package lentiviral RNA into the lentiviral particles (thepackaging vector). Preferably, the packaging vector contains a 5′ and 3′lentiviral LTR with the desired nucleic acid segment inserted betweenthem. The nucleic acid segment can be antisense molecules or morepreferably, encodes a protein such as an antibody. The packaging vectorpreferably contains a selectable marker gene. These are well known inthe art and include genes that change the sensitivity of a cell to astimulus such as a nutrient, an antibiotic, etc. Genes include those forneo (neomycin), puro (puromyicn), tk (thymidine kinase), multiple drugresistance (MDR), etc. Other genes express proteins that can readily bescreened for such as green fluorescent protein (GFP), blue fluorescentprotein (BFP), luciferase, LacZ, nerve growth factor receptor (NGFR),etc.

When an inducible promoter is used with the target molecule, minimalselection pressure is exerted on the transformed cells for those cellswhere the target molecule is “silenced”. Thus, identification of cellsdisplaying the marker also identifies cells that can express the targetmolecule. If an inducible promoter is not used, it is preferable to usea “forced-expression” system where the target molecule is linked to theselectable marker by use of an internal ribosome entry site (IRES) (seeMarasco et al., PCT/US96/16531).

IRES sequences are known in the art and include those fromencephalomycarditis virus (EMCV) (Ghattas, I. R. et al., 1991, Mol.Cell. Biol., 11: 5848-5849); BiP protein (Macejak and Sarnow, 1991,Nature, 353:91); the Antennapedia gene of Drosophila (exons d and e) (Ohet al., 1992, Genes & Dev., 6: 1643-1653); those in polio virus(Pelletier and Sonenberg, 1988, Nature 334:320325; see also Mountfordand Smith, 1985, TIG, 11:179-184). Preferably, the target molecule isoperably linked to an inducible promoter. Such systems allow the carefulregulation of gene expression. See Miller, N. and Whelan, J., 1997,Human Gene Therapy, 8: 803-815). Such systems include those using thelac repressor from E. coli as a transcription modulator to regulatetranscription from lac operator-bearing mammalian cell promoters (Brown,M. et al., 1987, Cell, 49:603-612) and those using the tetracyclinerepressor (tetR) (Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci.USA, 1992, 89:5547-5551; Yao, F. et al., 1998, Human Gene Therapy,9:1939-1950; Shockelt, P., et al., 1995, Proc. Natl. Acad. Sci. USA,92:6522-6526). Other systems include FK506 dimer, VP16 or p65 usingestradiol, RU486, diphenol murislerone or rapamycin [see Miller andWhelan, supra at FIG. 2]. Inducible systems are available fromINVITROGEN, CLONTECH and ARIAD. Systems using a repressor with theoperon are preferred. Regulation of transgene expression in target cellsrepresents a critical aspect of gene therapy. For example, a lacrepressor combined the tetracycline repressor (tetR) with thetranscription activator (VP16) can be used to create a tetR-mammaliancell transcription activator fusion protein, tTa (tetR-VP16), with thetetO-bearing minimal promoter derived from the human cytomegalovirus(hCMV) major immediate-early promoter to create a tetR-tet operatorsystem to control gene expression in mammalian cells. Recently Yao andcolleagues (F. Yao et al., Human Gene Therapy, supra] demonstrated thatthe tetracycline repressor (tetR) alone, rather than the tetR-mammaliancell transcription factor fusion derivatives can function as potenttrans-modulator to regulate gene expression in mammalian cells when thetetracycline operator is properly positioned downstream for the TATAelement of the CMVIE promoter. One particular advantage of thistetracycline inducible switch is that it does not require the use of atetracycline repressor-mammalian cells transactivator or repressorfusion protein, which in some instances can be toxic to cells (M. Gossenet al., 1992, Proc. Natl. Acad. Sci. USA, 89: 5547-5551; P. Shockett etal., 1995, Proc. Natl. Acad. Sci. USA, 92:6522-6526), to achieve itsregulatable effects. Preferably, the repressor is linked to the targetmolecule by an IRES sequence. Preferably, the inducible system is a tetRsystem. More preferably the system has the tetracycline operatordownstream of a promoter's TATA element such as with the CMVIE promoter.

The effectiveness of some inducible promoters increases over time. Insuch cases one can enhance the effectiveness of such systems byinserting multiple repressors in tandem, e.g. TetR linked to a TetR byan IRES. Alternatively, one can wait at least 3 days before screeningfor the desired function. While some silencing may occur, given thelarge number of cells being used, preferably at least 1×10⁴, morepreferably at least 1×10⁵, still more preferably at least 1×10⁶, andeven more preferably at least 1×10⁷, the effect of silencing is minimal.One can enhance expression of desired proteins by known means to enhancethe effectiveness of the system. For example, using the WoodchuckHepatitis Virus Port-transcriptional Regulatory Element (WPRC). See,Loeb, J. E., et al., 1999, Human Gene Therapy, 10:2295-2305; Zufferey,R., et al., 1999, J. of Virol., 73:2886-2892; Donello, J. E., et al.,1998, J. of Virol., 72:5085-5092).

In one embodiment, the stem cell cassette of the lentiviral genetransfer plasmid as described herein is flanked by LTRs and thePsi-sequence of HIV. The LTRs are necessary to integrate the therapeuticgene into the genome of the target cell, just as the LTRs in HIVintegrate the dsDNA copy of the virus into its host chromosome. ThePsi-sequence acts as a signal sequence and is necessary for packagingRNA with the reporter or therapeutic gene in virions. Viral proteinswhich make virus shells are provided in the packaging cell line, but arenot in context of the LTRs and Psi-sequences and so are not packagedinto virions. Thus, virus particles are produced that are replicationdeficient, so are designed to be unable to continue to infect their hostafter they deliver the coding sequences.

In one embodiment, the lentiviral vector described herein is flanked byloxP/Cre. Cre-lox allows site-specific recombination of DNA. There areother site-specific recombination sequences that can be used in ananalogous manner. This is a tool that researchers use to sitespecifically knockout or overexpress specific genes in mice. CRE is a 38kDa recombinase protein from bacteriophage P1 that mediatesintra-molecular and inter-molecular site-specific recombination betweenloxP sites. A loxP site consists of two 13 bp inverted repeats separatedby a 8 bp asymmetric spacer region. The detailed structure is givenbelow.

13 bp 8 bp 13 bp

ATAACTTCGTATA-GCATACAT-TATACGAAGTTAT (SEQ. ID. No. 25)

One molecule of CRE binds per inverted repeat or two CRE molecules lineup at one loxP site. The recombination occurs in the asymmetric spacerregion. Those 8 bases are also responsible for the directionality of thesite. Two loxP sequences in opposite orientation to each other invertthe intervening piece of DNA; two sites in direct orientation dictateexcision of the intervening DNA between the sites leaving one loxP sitebehind. This precise removal of DNA can be used to eliminate anendogenous gene or transgene (conditional gene deletion). The Cre/loxPsystem is a tool for tissue-specific (and, in connection with the tetsystem also time-specific) knockout or such genes that cannot beinvestigated in differentiated tissues because of their early embryoniclethality in mice with conventional knockouts. The Cre/loxP system canalso be used to activate a transgene. The Cre's DNA excising capabilitycan also be used to turn on a foreign gene by cutting out an interveningstop sequence between the promoter and the coding region of thetransgene. The Cre/loxP system is well known in the art, such as inZhongsen Li et al., Plant Molecular Biology, Vol. 65, No. 3, U.S. Pat.Nos. 5,919,676, 6,379,943, and 4,959,317 and these references are herebyincorporated by reference in their entirety. One of ordinary skill wouldbe able to construct a lentiviral vector with Cre/loxP.

Lentiviral vectors are usually created in a transient transfectionsystem in which a cell line is transfected with at least three separateplasmid expression systems. These include the transfer vector plasmid(portions of the HIV provirus), the packaging plasmid or construct, anda plasmid with the heterologous envelop gene (ENV) of a different virus.The three plasmid components of the vector are put into a packaging cellwhich is then inserted into the HIV shell. The virus portions of thevector contain insert sequences so that the virus cannot replicateinside the cell system.

The transfer vector plasmid contains cis-acting genetic sequencesnecessary for the vector to infect the target cell and for transfer ofthe genes (e.g. Oct4, Klf4, Sox2 and c-Myc) and contains restrictionsites for insertion of desired genes. The 3′ and 5′ LTRs, the originalenvelop proteins, and gag sequence promoter have been removed.

In some embodiments, the transfer gene plasmid is pHAGE-Tet-STEMCCAvector (SEQ. ID. No. 23) pHAGE-EF1α-STEMCCA vector (SEQ. ID. No. 24) andpHAGE-EF1α-STEMCCA-LoxP-RedLight (SEQ. ID. No. 50). In SEQ. ID. No. 50,the LoxP flanks the STEMCCA and the fourth gene in the cassette, c-Myc,has been replaced with a marker gene, mCherry, which codes for a redfluorescent protein.

In some embodiments, commercially available lentiviral transfer geneplasmid are used, e.g. pLenti4/V5-DEST™, pLenti6/V5-DEST™ or pLentivectors together with VIRAPOWER™ Lentiviral Expression systems fromInvitrogen. Further examples include but limited totetracycline-regulated replication-incompetent herpes simplex virusvectors described in F. Schmeisser, et al. (Human Gene Therapy, 2002,13: 2113-2124) and the LTRCMVR2, LTRAutoR2, TRECMVR2 and TREAutoR2lentiviral vectors described in D. Markusic et. al. (Nucleic AcidsResearch 2005 33(6):e63). The stem cell cassette can be constructed intothese plasmids.

In other embodiments, the transfer vector plasmids are plasmidsdescribed in U.S. Pat. Nos. 6,521,457 and 6,277,633. The stem cellcassette can be constructed into these plasmids.

The packaging plasmid is the backbone of the virus system. In thisplasmid are found the elements required for vector packaging such asstructural proteins, HIV genes (except the gene env which codes forinfection of T cells, or the vector would only be able to infect thesecells), and the enzymes that generate vector particles. Also containedis the human cytomegalovirus (hCMV) which is responsible for theexpression of the virus proteins during translation. The packagingsignals and their adjacent signals are removed so the parts responsiblefor packaging the viral DNA have been separated from the parts thatactivate them. Thus, the packaging sequences will not be incorporatedinto the viral genome and the virus will not reproduce after it hasinfected the host cell. Previous HIV vectors used two plasmids as thepackaging plasmid contained the viral envelop gene. However, in thenewer, better vectors the packaging plasmid lacks a viral envelop genebecause this has been shown to be more desirable in terms of titer(minimum volume needed to cause a particular result in titration),stability, and broad range of target cells.

The third plasmid's envelope gene of a different virus specifies whattype of cell to target and infect instead of the T cells. Normally HIVcan infect only helper T-cells because they use their gp120 protein tobind to the CD4 receptor. However, it is possible to geneticallyexchange the CD4 receptor-binding protein for another protein that codesfor the different cell type on which gene transfer will be performed.This gives the HIV lentiviral vector a broad range of possible targetcells. There are two types of heterologous envelope proteins. Theamphoteric envelop of MLV, another type of vector, is transcribed firstfollowed by the transcription of the G glycoproteins of the vesicularstomatitis virus, known as VSV-G. Both of these help to providestability to the vector by bringing together the particles that weremade by the packaging plasmid.

The lentiviral virion (particle) is expressed by a vector systemencoding the necessary viral proteins to produce a virion (viralparticle). Preferably, there is at least one vector containing a nucleicacid sequence encoding the lentiviral pol proteins necessary for reversetranscription and integration, operably linked to a promoter.Preferably, the pol proteins are expressed by multiple vectors. There isalso a vector containing a nucleic acid sequence encoding the lentiviralgag proteins necessary for forming a viral capsid operably linked to apromoter. Preferably, this gag nucleic acid sequence is on a separatevector than at least some of the pol nucleic acid sequence, still morepreferably it is on a separate vector from all the pol nucleic acidsequences that encode pol proteins.

Numerous modifications can be made to the vectors, which are used tocreate the particles to further minimize the chance of obtaining wildtype revertants. These include deletions of the U3 region of the LTR(for self inactivation), tat deletions and matrix (MA) deletions. Suchmodifications are well known in the art. One of ordinary skill in theart would be able to make these and similar modifications.

The gag, pol and env vector(s) do not contain nucleotides from thelentiviral genome that package lentiviral RNA, referred to as thelentiviral packaging sequence. In HIV this region corresponds to theregion between the 5′ major splice donor and the gag gene initiationcodon (nucleotides 301-319).

In some aspects, the vector(s) forming the particle preferably do notcontain a nucleic acid sequence from the lentiviral genome thatexpresses an envelope protein. Preferably, a separate vector thatcontains a nucleic acid sequence encoding an envelope protein operablylinked to a promoter is used. This env vector also does not contain alentiviral packaging sequence. In one embodiment the env nucleic acidsequence encodes a lentiviral envelope protein.

In another embodiment the envelope protein is not from the lentivirus,but from a different virus. The resultant particle is referred to as apseudotyped particle. By appropriate selection of envelopes one can“infect” virtually any cell. For example, one can use an env gene thatencodes an envelope protein that targets an endocytic compartment suchas that of the influenza virus, VSV-G, alpha viruses (Semliki forestvirus, Sindbis virus), arenaviruses (lymphocytic choriomeningitisvirus), flaviviruses (tick-borne encephalitis virus, Dengue virus),rhabdoviruses (vesicular stomatitis virus, rabies virus), andorthomyxoviruses (influenza virus). Other envelopes that can preferablybe used include those from Moloney Leukemia Virus such as MLV-E, MLV-Aand GALV. These latter envelopes are particularly preferred where thehost cell is a primary cell. Other envelope proteins can be selecteddepending upon the desired host cell. For example, targeting specificreceptors such as dopamine receptor for brain delivery. Another targetcan be vascular endothelium. These cells can be targeted using afilovirus envelope. For example, the GP of Ebola, which bypost-transcriptional modification become the GP1 and GP2 glycoproteins.In another embodiment, one can use different lentiviral capsids with apseudotyped envelope. For example, FIV or SHIV (U.S. Pat. No.5,654,195). A SHIV pseudotyped vector can readily be used in animalmodels such as monkeys.

The preferred lentivirus is a primate lentivirus (U.S. Pat. No.5,665,577) or a feline immunodeficiency virus (FIV) (Poeschla, E. M., etal., 1998, Nat. Medicine 4:354-357). The pol/gag nucleic acid segment(s)and the env nucleic acid segment will when expressed produce an emptylentiviral particle. By making the above-described modifications such asdeleting the tat coding region, the MA coding region, or the U3 regionof the LTR, the possibility of a reversion to a wild type virus has beenreduced to virtually nil.

In one embodiment, the lentiviral vector particle described herein isprepared according by a five-plasmid transfection procedure as describedin following protocol: Trans-IT 293 transfection for lentivirusproduction for cationic liposomal transfection.

Reagents:

-   -   293-T cells 90% confluent—pass the day before    -   Trans-IT 293 from Mirus cat#Mir2700    -   DMEM high glucose    -   Complete media (e.g. for 293T cells use 10% FBS in high glucose        DMEM with 1% pen/strep and 1×L-glutamine (5 cc from a 200 mM        stock)    -   DNA plasmids (backbone/insert, tat, rev, gag/pol, vsv-g)

DNA Proportions

transfer vector 20 1 1 1 2 backbone tat rev gag/pol vsv-g 30 ug 1.5 ug1.5 ug 1.5 ug 3 ug = 37.5 ug total DNAPrepare trans-IT/DNA/media mix: 2 ml DMEM per 15 cm plate and 3 (ul)volumes of trans-IT per 1 ug of DNA (e.g for 1×15 cm2 plate that willreceive 37.5 ug of DNA you need 3×37.5=112.5 ul of trans-IT in 2 ml ofDMEM and 37.5 ug of DNA.

Protocol:

-   -   1. prepare 293T cells the day before in 15 cm plates    -   2. Prepare DNA in an eppendorf tube by mixing together the 5        plasmids in the proportions above    -   3. Put amount of trans-IT needed into plain DMEM (2 ml DMEM per        15 cm plate). Put the trans-IT directly into the media! Don't        touch the walls of the container. Plastic de-activates the        reagent. Drop wise while vortexing and let stand at RT for 10        min.    -   4. Add 37.5 ug DNA plasmid mix to the 2 ml of trans-IT/DMEM,        drop wise while vortexing and let stand 15 min at RT.    -   5. Meanwhile take plate of 293T cells, aspirate off old media        and pour 13 cc of complete media (e.g 10% FCS DMEM, etc) into        each 15 cm plate.    -   6. Add the 2 ml of trans-IT/DNA/DMEM mix to each plate drop-wise    -   7. Mix gently by back and forth motion in two directions and        incubate at 37° C.    -   8. Start collecting supernatants 48 hr after transfection (this        is already the first collection, there is no need for washing),        replace with 15 ml complete media and collect every 12 hours        (4-5 collections). Use 0.45 um filter.    -   9. Concentrate by spinning for 1.5 hours at 16.5 k at 4° C.        Discard all the supernatant and let sit on ice for 2 hr.        Resuspend the virus in the liquid that came off the walls of the        tube (approx. 180 ul per tube).    -   10. Aliquot and store at −80° C.

In some embodiments, the lentiviral vector particle described herein isprepared according to any methods known in the art, for example, U.S.Pat. Nos. 6,428,953, 6,566,513, 6,613,569, 6,790,657, 7,226,780, and7,250,299. These references are hereby incorporated by reference intheir entirety.

In some embodiments that commercially available lentiviral vectors areused, e.g. pLenti4/V5-DEST™, pLenti6/V5-DEST™ or pLenti vectors, thelentiviral vector particles are produced using the recommendedlentiviral expression systems for that commercial lentiviral vector,e.g. with ViraPower™ Lentiviral Expression systems from Invitrogen.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in molecular biology can be found in The Merck Manual of Diagnosisand Therapy, 18th Edition, published by Merck Research Laboratories,2006 (ISBN 0-911910-18-2); Robert S. Porter et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8); The ELISA guidebook(Methods in molecular biology 149) by Crowther J. R. (2000);Fundamentals of RIA and Other Ligand Assays by Jeffrey Travis, 1979,Scientific Newsletters; Immunology by Werner Luttmann, published byElsevier, 2006. Definitions of common terms in molecular biology canalso be found in Benjamin Lewin, Genes IX, published by Jones & BartlettPublishing, 2007 (ISBN-13: 9780763740634); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., MolecularCloning: A Laboratory Manual (2 ed.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1989); Davis et al., Basic Methodsin Molecular Biology, Elsevier Science Publishing, Inc., New York, USA(1986); or Methods in Enzymology: Guide to Molecular Cloning TechniquesVol. 152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., SanDiego, USA (1987)), Current Protocols in Molecular Biology (CPMB) (FredM. Ausubel, et al. ed., John Wiley and Sons, Inc.), Current Protocols inProtein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley andSons, Inc.) and Current Protocols in Immunology (CPI) (John E. Coligan,et. al., ed. John Wiley and Sons, Inc.), Current Protocols in CellBiology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons,Inc.), Culture of Animal Cells: A Manual of Basic Technique by R. IanFreshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell CultureMethods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and DavidBarnes editors, Academic Press, 1st edition, 1998) which are allincorporated by reference herein in their entireties.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean±1%.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. The term“comprises” means “includes.” The abbreviation, “e.g.” is derived fromthe Latin exempli gratia, and is used herein to indicate a non-limitingexample. Thus, the abbreviation “e.g.” is synonymous with the term “forexample.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Application

In one embodiment, the present also provides a method of reprogramming asomatic cell; the method comprising contacting a somatic cell with alentiviral vector described herein.

In one embodiment, the somatic cell is a mammalian cell. In oneembodiment, the somatic cell is a mammalian cell derived from internalorgans-heart, kidney, liver, lungs, bladder, intestines; skin, bones,blood, cartilage and connective tissues. One skilled in the art would beable to isolate somatic cells from an individual, culture expand thecells and infect these cells with a lentiviral vector particle describedherein in vitro. The infected cells can then be analyzed for expressionof embryonic stem (ES) cell markers, e.g. SSEAs, the transgenetranscription factors such as OCT4, KLF4, SOX2, and c-MYC, or markergenes such as mCherry used herein etc, and for teratoma formation asdescribed herein for confirmation of reprogramming of the somatic cellinto a stem-cell-like cell.

In one embodiment, the method of programming somatic cells furthercomprises excising the integrated lentiviral vector after the infectedcells demonstrate expression of embryonic stem (ES) cell markers, e.g.SSEAs, the transgene transcription factors such as OCT4, KLF4, SOX2, andc-MYC, or marker genes such as mCherry used herein. In one embodiment,the infected cells that have expressed embryonic stem (ES) cell markers,e.g. SSEAs, the transgene transcription factors such as OCT4, KLF4,SOX2, and c-MYC, or marker genes such as mCherry used herein are furtherinfected with a defective Adenoviral vector carrying the Cre recombinasegene (Adeno-Cre). The excision is performed with a Cre recombinase. Inone embodiment, the Cre is an inducible Cre, e.g. Cre-ERT2 which isinduced by tamoxifen.

The present invention can be defined by any of the followingalphabetized paragraphs:

-   -   [A] A lentiviral vector particle comprising a nucleic acid        sequence comprising a sequence encoding: (a) a first gene; (b) a        second gene; (c) a third gene; (d) an optional fourth gene,        wherein the first, second, third and optional fourth genes are        selected from the group consisting of Oct4, Klf4, Sox2, cMyc,        Lin28, and Nanog, and wherein the first, second, third and        optional fourth genes are not identical; (e) a first        ‘self-cleaving’ 2A peptide; (f) a second ‘self-cleaving’ 2A        peptide; and (g) an internal ribosome entry site (IRES); wherein        the nucleic acid sequence is operably linked to a promoter,        wherein the sequences encoding the first, second, third and        optional fourth genes, first and second ‘self-cleaving’ 2A        peptides, and the IRES are transcribed from the promoter as a        multi-cistronic RNA.    -   [B] The lentiviral vector particle of paragraph [A], wherein a        marker gene can be included, wherein the marker gene encodes an        optically visible protein or an enzyme.    -   [C] The lentiviral vector particle of paragraph [A] or [B],        wherein the sequences encoding the first, second, third and        optional fourth genes are arranged in tandem, wherein the genes        are oriented in the sense direction, and wherein the genes are        arranged in any order.    -   [D] The lentiviral vector particle of any of paragraphs [A]-[C],        wherein the sequence encoding internal ribosome entry site        (IRES) is between the second and third genes in the tandem        arrangement of the first, second, third and optional fourth        genes.    -   [E] The lentiviral vector particle of any of paragraphs [A]-[D],        wherein the sequence encoding the first ‘self-cleaving’ 2A        peptide is between the first and second genes in the tandem        arrangement of the first, second, third and optional fourth        genes.    -   [F] The lentiviral vector particle of any of paragraphs [A]-[E],        wherein the sequence encoding the second ‘self-cleaving’ 2A        peptide is between the third and optional fourth genes in the        tandem arrangement of the first, second, third and optional        fourth genes.    -   [G] The lentiviral vector particle of paragraph [E], wherein the        first ‘self-cleaving’ 2A peptide is selected from the group        consisting of F2A, E2A, T2A and P2A.    -   [H] The lentiviral vector particle of paragraph [F] wherein the        second ‘self-cleaving’ 2A peptide is selected from the group        consisting of F2A, E2A, T2A and P2A.    -   [I] The lentiviral vector particle of any of paragraphs [A]-[H],        wherein the second ‘self-cleaving’ 2A peptide is different from        the first ‘self-cleaving’ 2A peptide.    -   [J] The lentiviral vector particle of any of paragraphs [A]-[I],        wherein the promoter is inducible.    -   [K] The lentiviral vector particle of any of paragraphs [A]-[J],        wherein the promoter is constitutive.    -   [L] The lentiviral vector particle of paragraph [J], wherein the        promoter is tetracycline regulated.    -   [M] The lentiviral vector particle of paragraph [K], wherein the        promoter is EF-1alpha.    -   [N] A lentiviral vector particle comprising a nucleic acid        sequence comprising a sequence encoding: (a) a Oct4 gene; (b) a        Klf4 gene; (c) a Sox2 gene; (d) a c-Myc gene; (e) a first        ‘self-cleaving’ 2A peptide; (f) a second ‘self-cleaving’ 2A        peptide; and (g) an internal ribosome entry site (IRES); wherein        the nucleic acid sequence is operably linked to a promoter,        wherein the sequences encoding Oct4, Klf4, Sox2, c-Myc, first        and second ‘self-cleaving’ 2A peptides, and the IRES are        transcribed from the promoter as a multi-cistronic RNA.

[O] The lentiviral vector particle of paragraph [N], wherein thesequences encoding the four genes: Oct4, Klf4, Sox2, and c-Myc, arearranged in tandem, wherein the genes are oriented in the sensedirection, and wherein the genes are arranged in any order.

-   -   [P] The lentiviral vector particle of paragraph [N] or [O],        wherein the sequence encoding internal ribosome entry site        (IRES) is between the second and third genes in the tandem        arrangement of the four genes: Oct4, Klf4, Sox2, and c-Myc.    -   [Q] The lentiviral vector particle of any of paragraphs [N]-[P]        wherein the sequence encoding the first ‘self-cleaving’ 2A        peptide is between the first and second genes in the tandem        arrangement of the four genes: Oct4, Klf4, Sox2, and c-Myc.    -   [R] The lentiviral vector particle of any of paragraphs [N]-[Q],        wherein the sequence encoding the second ‘self-cleaving’ 2A        peptide is between the third and forth genes in the tandem        arrangement of the four genes: Oct4, Klf4, Sox2, and c-Myc.    -   [S] The lentiviral vector particle of paragraph [Q], wherein the        first ‘self-cleaving’ 2A peptide is selected from the group        consisting of F2A, E2A, T2A and P2A.    -   [T] The lentiviral vector particle of paragraph [R] wherein the        second ‘self-cleaving’ 2A peptide is selected from the group        consisting of F2A, E2A, T2A and P2A.    -   [U] The lentiviral vector particle of any of paragraphs [N]-[T],        wherein the second ‘self-cleaving’ 2A peptide is different from        the first ‘self-cleaving’ 2A peptide.    -   [V] The lentiviral vector particle of any of paragraphs [N]-[U],        wherein the promoter is inducible.    -   [W] The lentiviral vector particle of any of paragraphs [N]-[U],        wherein the promoter is constitutive.    -   [X] The lentiviral vector particle of paragraph [V], wherein the        promoter is tetracycline regulated.    -   [Y] The lentiviral vector particle of paragraph [W], wherein the        promoter is EF-1alpha.    -   [Z] A lentiviral vector particle capable for reprogramming a        somatic cell to a stem-cell-like cell, the vector particle        comprising a nucleic acid sequence comprising a sequence        encoding: (a) a Oct4 gene; (b) a Klf4 gene; (c) a Sox2 gene; (d)        a marker gene; (e) a first ‘self-cleaving’ 2A peptide; (f) a        second ‘self-cleaving’ 2A peptide; and (g) an internal ribosome        entry site (IRES), wherein the nucleic acid sequence is operably        linked to a promoter, and wherein the sequences encoding Oct4,        Klf4, Sox2, the marker gene, first and second ‘self-cleaving’ 2A        peptides, and the IRES are transcribed from the promoter as a        multi-cistronic RNA.    -   [AA] The lentiviral vector particle of paragraph [Z], wherein        the marker gene encodes an optically visible protein or an        enzyme.    -   [BB] The lentiviral vector particle of paragraph [Z] or [AA],        wherein the sequences encoding the four genes: Oct4, Klf4, Sox2,        and the marker gene, are arranged in tandem, wherein the genes        are oriented in the sense direction, and wherein the genes are        arranged in any order.    -   [CC] The lentiviral vector particle of any of paragraphs        [Z]-[BB], wherein the sequence encoding internal ribosome entry        site (IRES) is between the second and third genes in the tandem        arrangement of the four genes: Oct4, Klf4, Sox2, and the marker        gene.    -   [DD] The lentiviral vector particle of any of paragraphs        [Z]-[CC], wherein the sequence encoding the first        ‘self-cleaving’ 2A peptide is between the first and second genes        in the tandem arrangement of the four genes: Oct4, Klf4, Sox2,        and the marker gene.    -   [EE] The lentiviral vector particle of any of paragraphs        [Z]-[DD], wherein the sequence encoding the second        ‘self-cleaving’ 2A peptide is between the third and forth genes        in the tandem arrangement of the four genes: Oct4, Klf4, Sox2,        and the marker gene.    -   [FF] The lentiviral vector particle of any of paragraphs        [A]-[EE] further comprises a Cre-LoxP excision sequence.    -   [GG] A vector system comprising: (a) a first vector containing a        lentiviral gag gene encoding a lentiviral Gag protein, wherein        the lentiviral gag gene is operably linked to a promoter and a        polyadenylation sequence; (b) a second vector containing an env        gene encoding a functional Env protein, wherein the env gene is        operably linked to a promoter and a polyadenylation        sequence; (c) a lentiviral pol gene encoding a lentiviral Pol        protein, wherein the pol protein is at least an integrase, and        the pol gene is on the first or second vectors or on at least a        third vector, wherein the lentiviral pol gene is operably linked        to a promoter and a polyadenylation sequence, wherein the at        least first, second and third vectors do not contain sufficient        nucleotides to encode the lentiviral Gag and Pol and the Env        protein on a single vector, wherein the vectors do not contain        nucleotides of the lentiviral genome referred to as a packaging        segment to effectively package lentiviral RNA, and wherein the        lentiviral proteins and the Env protein when expressed in        combination form a lentivirus virion containing an Env protein        around a lentiviral capsid; and (d) a packaging gene transfer        plasmid comprising a stem cell cassette nucleic acid sequence        encoding: a first gene; a second gene; a third gene; an optional        fourth gene; a first ‘self-cleaving’ 2A peptide; a second        ‘self-cleaving’ 2A peptide; and an internal ribosome entry site        (IRES); wherein the first, second, third and optional fourth        genes are selected from the group consisting of Oct4, Klf4,        Sox2, c-Myc, Lin28, and Nanog; wherein the first, second, third        and optional fourth genes are not identical; wherein if the        optional fourth gene is not selected from the group consisting        of Oct4, Klf4, Sox2, c-Myc, Lin28, and Nanog, a marker gene is        included in its place, wherein the marker gene encodes an        optically visible protein or an enzyme; wherein the nucleic acid        sequence is operably linked to a promoter, and wherein the        sequences encoding the first gene; the second gene; the third        gene; the optional fourth gene, first and second ‘self-cleaving’        2A peptides, and the IRES are transcribed from the promoter as a        multi-cistronic RNA.    -   [HH] The vector system of paragraph [GG], wherein the integrase        has been modified so that it is not capable of integration.    -   [II] The vector system of paragraph [GG] or [HH], wherein the        lentivirus is selected from the group consisting of HIV, HIV-2,        FIV, and SIV.    -   [JJ] The vector system of paragraph [GG], [HH] or [II], wherein        the env gene encodes an envelope from a different virus, and is        of a different source from the gag and pol genes.    -   [KK] The vector system of any of paragraphs [GG]-[JJ], wherein        the sequences encoding the first, second, third and optional        fourth genes are arranged in tandem, wherein the genes are        oriented in the sense direction, and wherein the genes are        arranged in any order.    -   [LL] The vector system of any of paragraphs [GG]-[KK], wherein        the sequence encoding internal ribosome entry site (IRES) is        between the second and third genes in the tandem arrangement of        the first, second, third and optional fourth genes.    -   [MM] The vector system of any of paragraphs [GG]-[LL], wherein        the sequence encoding the first ‘self-cleaving’ 2A peptide is        between the first and second genes in the tandem arrangement of        the first, second, third and optional fourth genes.    -   [NN] The vector system of any of paragraphs [GG]-[MM], wherein        the sequence encoding the second ‘self-cleaving’ 2A peptide is        between the third and forth genes in the tandem arrangement of        the first, second, third and optional fourth genes.    -   [OO] The vector system of paragraph [MM], wherein the first        ‘self-cleaving’ 2A peptide is selected from the group consisting        of F2A, E2A, T2A and P2A.    -   [PP] The vector system of paragraph [NN], wherein the second        ‘self-cleaving’ 2A peptide is selected from the group consisting        of F2A, E2A, T2A and P2A.    -   [QQ] The vector system of any of paragraphs [GG]-[PP], wherein        the second ‘self-cleaving’ 2A peptide is different from the        first ‘self-cleaving’ 2A peptide.    -   [RR] The vector system of any of paragraphs [GG]-[QQ], wherein        the promoter is inducible.    -   [SS] The vector system of any of paragraphs [GG]-[QQ], wherein        the promoter is constitutive.    -   [TT] The vector system of paragraph [RR], wherein the promoter        is tetracycline regulated.    -   [UU] The vector system of paragraph [SS], wherein the promoter        is EF-1alpha.    -   [VV] A vector system comprising: (a) a first vector containing a        lentiviral gag gene encoding a lentiviral Gag protein, wherein        the lentiviral gag gene is operably linked to a promoter and a        polyadenylation sequence, (b) a second vector containing an env        gene encoding a functional envelope protein, wherein the env        gene is operably linked to a promoter and a polyadenylation        sequence; (c) a lentiviral pol gene encoding a lentiviral Pol        protein, wherein the pol protein is at least an integrase, and        the pol gene is on the first or second vectors or on at least a        third vector, wherein the lentiviral pol gene is operably linked        to a promoter and a polyadenylation sequence; wherein the at        least first, second and third vectors do not contain sufficient        nucleotides to encode the lentiviral Gag and Pol and the        envelope protein on a single vector; and wherein the vectors do        not contain nucleotides of the lentiviral genome referred to as        a packaging segment to effectively package lentiviral RNA; and        wherein the lentiviral proteins and the envelope protein when        expressed in combination form a lentivirus virion containing an        envelope protein around a lentiviral capsid; and (d) a packaging        gene transfer plasmid comprising a stem cell cassette nucleic        acid sequence encoding: a Oct4 gene; a Klf4 gene; a Sox2 gene; a        c-Myc gene; a first ‘self-cleaving’ 2A peptide; a second        ‘self-cleaving’ 2A peptide; an internal ribosome entry site        (IRES), wherein the nucleic acid sequence is operably linked to        a promoter, wherein the sequences encoding Oct4, Klf4, Sox2,        c-Myc, first and second ‘self-cleaving’ 2A peptides, and the        IRES are transcribed from the promoter as a multi-cistronic RNA.    -   [WW] The vector system of paragraph [VV], wherein the integrase        has been modified so that it is not capable of integration.    -   [XX] The vector system of paragraph [VV] or [WW], wherein the        lentivirus is selected from the group consisting of HIV, HIV-2,        FIV, and SIV.    -   [YY] The vector system of paragraph [VV], [WW] or [XX], wherein        the env gene encodes an envelope from a different virus, and is        of a different source from the gag and pol genes.    -   [ZZ] The vector system of any of paragraphs [VV]-[YY], wherein        the sequences encoding the four genes: Oct4, Klf4, Sox2, and        c-Myc, are arranged in tandem, wherein the genes are oriented in        the sense direction, and wherein the genes are arranged in any        order.    -   [AAA] The vector system of any of paragraphs [VV]-[ZZ], wherein        the sequence encoding internal ribosome entry site (IRES) is        between the second and third genes in the tandem arrangement of        the four genes: Oct4, Klf4, Sox2, and c-Myc.    -   [BBB] The vector system of any of paragraphs [VV]-[AAA], wherein        the sequence encoding the first ‘self-cleaving’ 2A peptide is        between the first and second genes in the tandem arrangement the        four genes: Oct4, Klf4, Sox2, and c-Myc.    -   [CCC] The vector system of any of paragraphs [VV]-[BBB], wherein        the sequence encoding the second ‘self-cleaving’ 2A peptide is        between the third and forth genes in the tandem arrangement the        four genes: Oct4, Klf4, Sox2, and c-Myc.    -   [DDD] The vector system of any of paragraphs [GG]-[CCC], wherein        the sequence encoding cMyc is replaced by a marker gene, wherein        the marker gene encodes an optically visible protein or an        enzyme.    -   [EEE] The vector system of any of paragraphs [GG]-[DDD] further        comprises a Cre-LoxP excision sequence.    -   [FFF] A method of reprogramming a somatic cell; the method        comprising contacting a somatic cell with a lentiviral vector of        any of paragraphs [A]-[EEE].    -   [GGG] An induced pluripotent stem cell derived by transduction        of any lentiviral vector of any paragraphs [A]-[GGG].

This invention is further illustrated by the following example whichshould not be construed as limiting. The contents of all referencescited throughout this application, as well as the figures and table areincorporated herein by reference.

EXAMPLES Experimental Procedures Construction of Lentiviral Vectors

A multiple expression system was designed based on the pHAGE lentiviralvector. pHAGE is a 3^(rd) generation lentiviral vector previouslydescribed (Mostoslaysky, G., et. al. 2006, Proc. Natl. Acad. Sci. USA103:16406-16411). The pHAGE was re-engineered for multicistronic geneexpression to accomplish the production of the proteins Oct4, Klf4, Sox2and c-Myc from a single transcript. The templates used are Oct4: GenbankAccession No. NM_(—)013633; Sox2: Genbank Accession No. NM_(—)011443;Klf4: Genbank Accession No. NM_(—)010637; cMyc: Genbank Accession No.NM_(—)010849. First, two DNA fragments were generated by overlapping PCRusing Pfu TURBO® DNA polymerase (STRATAGENE®); one fragment consistingof the complementary DNAs (cDNAs) of murine Oct4 and Klf4 separated byan intervening sequence encoding the F2A peptide, the second fragmentcontaining the cDNAs of murine Sox2 and c-Myc, separated by anintervening sequence encoding the E2A peptide. To obtain theOct4-F2A-Klf4 fragment, two PCR reactions were carried out using theprimer pairs Oct4 5′ NotI/Oct4-F2A 3′ and F2A-Klf4 5′/Klf4 3′ BglII (seeTable 1) under the following conditions: initial denaturation at 94° C.for 2 min followed by 35 cycles of 45 s at 94° C., 45 s at 60° C. and 2min at 72° C. Aliquots of the two purified amplicons were then mixed ina 1:1 ratio and used in a second PCR round with the primers Oct4 5′ NotIand Klf4 3′ BamHI under the following conditions: initial denaturationat 94° C. for 2 min, 5 cycles of 45 s at 94° C., 45 s at 58° C. and 2min at 72° C., and 30 cycles of 45 s at 94° C., 45 s at 62° C. and 2 minat 72° C. The resulting fragment (Oct4-F2A-Klf4) was gel-purified andinserted by directional cloning into the Not I- and BamH I-digestedpHAGE2 lentiviral vector backbone upstream of an IRES element.Similarly, a DNA fragment corresponding to Sox2-E2A-cMyc was obtained byPCR using the conditions described above and the primer pairs Sox2 5′NdeI/Sox2-E2A 3′ and E2A-cMyc 5′/cMyc 3′ ClaI (first round ofamplification) and Sox2 5′ NdeI/c-Myc 3′ ClaI (second round ofamplification). This fragment (Sox2-E2A-cMyc) was then inserted betweenthe NdeI and ClaI sites, downstream of the IRES element of thepHAGE2-Oct4-F2A-Klf4 vector. Finally, the human EF1α promoter or theTetO/miniCMV promoter was cloned into SpeI and NotI sites of therecombinant vector to generate pHAGE-EF1a-STEMCCA and pHAGE-Tet-STEMCCAvectors, respectively. Sequence identity was confirmed by sequencing.

TABLE 1 Primers used for vector construction. Oct4 5′ NotICACCGGCGGCCGCCATGGATCCTCGAACCTGGCTAAGCTTC CAAG (SEQ. ID. No. 15)Oct4-F2A 3′ CTTGAGAAGGTCAAAATTCAAAGTCTGTTTCACGCCACTTCCGTTTGAATGCATGGGAGAGCCCAGAGCAG (SEQ. ID. No. 16) F2A-Klf4 5′AAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCCATGGCTAGCGACGCTCTGC TCCC (SEQ. ID. No. 17) Klf4 3′TTTGGATCCTTAAAAGTGCCTCTTCATGTGTAAGGCAAG BamHI (SEQ. ID. No. 18) Sox25′ NdeI GGTTTCTTACATATGATGTATAACATGATGGAGACGGAGCT GAAG (SEQ. ID. No. 19)Sox2-E2A 3′ TTTCAACATCGCCAGCGAGTTTCAACAAAGCGTAGTTAGTACATTGCCCACTACCCATGTGCGACAGGGGCAGTGTGCCGTT AATGGCCG (SEQ. ID. No. 20)E2A-cMyc 5′ CTTTGTTGAAACTCGCTGGCGATGTTGAAAGTAACCCCGGTCCTATGCCCCTCAACGTGAACTTCACCAACAGGAACTATG (SEQ. ID. No. 21) cMyc 3′ ClaIGGTTTATCGATTTATGCACCAGAGTTTCGAAGCTGTTC (SEQ. ID. No. 22)

In order to obtain a floxed version of STEMCCA after viral integration,that would enable Cre-mediated excision of the vector followingreprogramming, a 34-bp loxP site was inserted in the 3′ dU3 LTR regionof EF 1-STEMCCA. During reverse transcription, the loxP sequence iscopied to the 5′ LTR resulting in a vector flanked by two loxP sites.

Two versions of the constitutive EF1-STEMCCA vector described above wereengineered, expressing either 4 reprogramming transcription factors(STEMCCA-loxP) or 3 reprogramming transcription factors plus mCherry(STEMCCA-loxP-RedLight). A loxP site was introduced within the U3 regionof the 3′ LTR. Upon formation of the provirus a floxed version of eachSTEMCCA vector is produced. Following exposure to Cre, the entirecassette is excised. LTR: long terminal repeat; PSI: packaging signal;RRE: rev responsive element; cpPu: central polypuryne tract; WPRE:Woodchuck hepatitis virus post-transcriptional regulatory element; dU3:deleted U3.

Cell Culture

Tail-tip fibroblasts (TTFs) were derived from Sox2-GFP/R26-M2rtTA doubleknock-in mice (Stadtfeld, M., et. al., Cell Stem Cell 2:230-240) Thesecells carry an M2rtTA gene encoding a reverse tetracyclinetransactivator targeted to the constitutively active ROSA26 locus aswell as a reporter cDNA targeted to the Sox2 locus. Tail snips from 3-4day old mice were cultured according to standard methods to expand TTFsin fibroblast growth media (DMEM 10% FBS, L-Glutamine,penicillin/streptomycin). TTFs were infected at passage 3 for generationof iPS cells.

Lentivirus Production and Infection

Lentiviruses were produced using a five plasmid transfection system in293T packaging cells as previously described (Mostoslaysky, G., et. al.2006, Proc. Natl. Acad. Sci. USA 103:16406-16411). Generation oflentiviral vectors was accomplished by a five-plasmid transfectionprocedure. 293T cells were transfected using TransIT 293 (Minis,Madison, Wis.) according to the manufacturer instructions with thebackbone pHAGE vector together with four expression vectors encoding thepackaging proteins gagpol, rev, tat and the G-protein of the vesicularstomatitis virus (VSV). The gagpol helper plasmid has beencodon-optimized for efficient mammalian expression and modified toseverely reduce the homology with the gag sequences present in thevector packaging signal. In addition, it makes the gagpol expressionrev-independent. All of the expression helper plasmids contain only thecoding sequences, with minimal 5′ or 3′ untranslated sequences and nointrons. In addition, the backbone contains the Woodchuck Hepatitisvirus post-transcriptional regulatory element (WPRE), and the centralpolypurine tract (cppt) to enhance levels of transcription and geneexpression. Viral supernatants were collected starting 24 hr aftertransfection, for four consecutive times every twelve hours, pooled andfiltered through a 0.45 mm filter. Viral supernantants were thenconcentrated ˜100 fold by ultracentrifugation in a Beckman centrifuge,for 1.5 hr at 16500 rpm and stored at −80° C.

Supernatants were collected every 12 hours during two consecutive daysstarting 48 hours after transfection and viral particles wereconcentrated by centrifugation at 16,500 rpm for 1.5 hours at 4° C.Approximately 100,000 fibroblasts were seeded on plastic in 35-mmculture plates and infected with 15-20 ml of concentrated virus in thepresence of polybrene (51 g/ml). The media was replaced after 16 hourswith mouse ES cell media (DMEM supplemented with 15% FBS, L-glutamine,penicillin/streptomycin, nonessential amino acids, (3-mercaptoethanoland 1000 U/ml LIF) and changed every 2-3 days. Doxycycline(Sigma-Aldrich) was added at a final concentration of 1 μg/ml, whereindicated, and removed at day 10 post-infection. iPS colonies werepicked 20 to 25 days post-infection based on morphology and GFPexpression and expanded by plating on Mitomycin C treated MEFs in EScell media.

iPS colonies were mechanically isolated 15 to 20 days post-infectionwith STEMCCA-loxP or 25-30 days post-infection withSTEMCCA-loxP-RedLight based on morphology and expanded by plating onMitomycin C treated MEFs in ES cell media.

Infection of iPS Cells with Adeno-Cre

Excision of STEMCCA was performed by infecting iPS cells with adefective adenoviral vector expressing Cre-recombinase (Adeno-Cre), akind gift of Jeng-Shin Lee and Richard C. Mulligan from the Harvard GeneTherapy Initiative. The recombinant adenovirus was propagated in 293cells, purified by CsCl gradient centrifugation and desalted on aSephadex G-50 column (GE Healthcare UK Limited, Little Chalfont,Buckinghamshire, U.K.) in PBS/3% glycerol. For Adeno-Cre infection, iPScells were tripsinized and washed once with PBS. Approximately 100,000cells in 100 μl of ESC media were mixed with 3 μl of Adeno-Cre in amicrofuge tube and incubated for 6 hours at 37° C. in a 5% CO₂incubator. Cells were then washed with PBS, seeded on Mitomycin Ctreated MEFs and cultured in ESC media until colonies appeared. For eachiPS clone infected with Adeno-Cre, several subclones were isolated andexpanded as described above. Finally, the efficiency of Cre-recombinaseactivity was assessed by PCR and Southern Blot or by mCherry redfluorescence, as indicated in the text.

Antibodies

Immunofluorescence and western blot assays were performed using routinemethods. The following primary antibodies were used: rabbit anti-Oct4(Abcam), goat anti-Klf4 (R&D Systems), mouse anti-Sox2 (R&D Systems),mouse anti-cMyc (NeoMarkers), mouse anti-GAPDH (Millipore) and mouseanti-SSEA-1 (Santa Cruz Biotechnology). The followingfluorochrome-conjugated secondary antibodies were applied: Alexa Fluor488 donkey anti-goat, Texas Red goat anti-rabbit and Cy3 goat anti-mouse(Molecular Probes). Alkaline phosphatase staining was performed with theVector Red Substrate Kit (Vector Laboratories) according to themanufacturer's instructions. Flow cytometry was performed using standardprocedures. All flow cytometric data were acquired using equipmentmaintained by the Boston University Medical Campus Flow Cytometry CoreFacility.

RT-PCR of Marker Genes

Total RNA was purified with TriPure Isolation Reagent (ROCHE®). Onemicrogram of RNA was reverse-transcribed using ImProm-II ReverseTranscriptase (PROMEGA®) according to the manufacturer's instructions.Primers for ES cell marker genes are described elsewhere (Takahashi andYamanaka, 2006, Cell 126, 663-676). The primers for ES cell markergenes, and names of the marker genes are listed below:

Oct3/4 (Pou5f1): TCT TTC CAC CAG GCC CCC GGC TC); TGC GGG CGG ACA TGGGGA GAT CC (SEQ ID NOS 26 and 53, respectively, in order of appearance)Fgf4: CGT GGT GAG CAT CTT CGG AGT GG; CCT TCT TGG TCC GCC CGT TCT TA(SEQ ID NOS 27 and 54, respectively, in order of appearance)Nanog: CAG GTG TTT GAG GGT AGC TC; CGG TTC ATC ATG GTA CAG TC (SEQ IDNOS 28 and 55, respectively, in order of appearance)Rex1 (Zfp42): ACG AGT GGC AGT TTC TTC TTG GGA; TAT GAC TCA CTT CCA GGGGGC ACT (SEQ ID NOS 29 and 56, respectively, in order of appearance)Esg1 (Dppa5): GAA GTC TGG TTC CTT GGC AGG ATG; ACT CGA TAC ACT GGC CTAGC (SEQ ID NOS 30 and 57, respectively, in order of appearance)Gdf3: GTT CCA ACC TGT GCC TCG CGT CTT; AGC GAG GCA TGG AGA GAG CGG AGCAG (SEQ ID NOS 31 and 58, respectively, in order of appearance)Ecat1: TGT GGG GCC CTG AAA GGC GAG CTG AGA T; ATG GGC CGC CAT ACG ACGACG CTC AAC T (SEQ ID NOS 32 and 59, respectively, in order ofappearance)Dax1: TGC TGC GGT CCA GGC CAT CAA GAG; GGG CAC TGT TCA GTT CAG CGG ATC(SEQ ID NOS 33 and 60, respectively, in order of appearance)Zfp296: CCA TTA GGG GCC ATC ATC GCT TTC; CAC TGC TCA CTG GAG GGG GCT TGC(SEQ ID NOS 34 and 61, respectively, in order of appearance)Cripto: ATG GAC GCA ACT GTG AAC ATG ATG TTC GCA; CTT TGA GGT CCT GGT CCATCA CGT GAC CAT (SEQ ID NOS 35 and 62, respectively, in order ofappearance)Nat1: ATT CTT CGT TGT CAA GCC GCC AAA GTG GAG; AGT TGT TTG CTG CGG AGTTGT CAT CTC GTC (SEQ ID NOS 36 and 63, respectively, in order ofappearance)

Detection of the proviral DNA by PCR was carried out using 50 ng ofgenomic DNA and the primers endo-MycS (5′-ACGAGCACAAGCTCACCTCT-3′ (SEQ.ID. No. 37)) and A-WPRE (5′-TCAGCAAACACAGTGCACACC-3′ (SEQ. ID. No. 38)).PCR reactions consisted of 30 cycles of 95° C. for 30 seconds, 65° C.for 45 seconds and 72° C. for 45 seconds.

To create a LoxP site, complementary 5′-phosphorilated oligonucleotidescontaining a loxP sequence flanked by overhang sites for AscI(5′-/5Phos/CGCGCAGGTACCATAACTTCGTATAATGTATGCTATACGAAGTTATGG-3′ (SEQ. ID.No. 39)) and5′-/5Phos/CGCGCCATAACTTCGTATAGCATACATTATACGAAGTTATGGTACCTG-3′ (SEQ. ID.No. 40)) were annealed and ligated to the vector previously digestedwith the same enzyme to create STEMCCA-loxP. The STEMCCA-loxP-RedLightvector was constructed as follows. A PCR product consisting of themurine Sox2 gene and the mCherry gene separated by an interveningsequence encoding the E2A peptide was generated by overlapping PCR usingPfu TURBO® DNA polymerase (STRATAGENE®). In brief, two PCR reactionswere carried out with primer pairs Sox2 5′NdeI/Sox2-E2A 3′ andE2A-mCherry 5′/mCherry 3′ClaI under the following conditions: initialdenaturation at 95° C. for 2 min followed by 35 cycles of 45 s at 95°C., 45 s at 60° C. and 2 min at 72° C. Aliquots of the two purifiedamplicons were then mixed in a 1:1 ratio and used in a second PCR roundwith the primers Sox2 5 ‘NdeI and mCherry 3’ ClaI under the followingconditions: initial denaturation at 95° C. for 2 min, 5 cycles of 45 sat 95° C., 45 s at 58° C. and 2 min at 72° C., and 30 cycles of 45 s at95° C., 45 s at 62° C. and 2 min at 72° C. The resulting fragmentSox2-E2A-mCherry was gel-purified and inserted by directional cloninginto the Nde I- and Cla I-digested STEMCCA-loxP vector. Sequenceidentity was confirmed by sequencing. Primer sequences were as follows:Sox2 5′ NdeI (5′-GGTTTCTTACATATGATGTATAACATGATGGAGACGGAGCTGAAG-3′ (SEQ.ID. No. 19)), Sox2-E2A 3′(5′-TTTCAACATCGCCAGCGAGTTTCAACAAAGCGTAGTTAGTACATTGCCCACTACCCATGTGCGACAGGGGCAGTGTGCCGTTAATGGCCG-3′ SEQ. ID. No. 20), E2A-mCherry 5′(5′-CTTTGTTGAAACTCGCTGGCGATGTTGAAAGTAACCCCGGTCCTATGGTGAGCAAGGGCGAGGAGGATAACATGGCC-3′ SEQ. ID. No. 41), mCherry 3′ ClaI(5′-ATCGATTTACTTGTACAGCTCGTCCATGCCGCCGGTG-3′ SEQ. ID. No. 42).

Quantitative RT-PCR

qRT-PCR for STEMCCA transcript was carried out in a StepOnePlusreal-time PCR system (APPLIED BIOSYSTEMS®) using Taqman custom primersand probe specific to the genes of interest as described by themanufacturer. Amplification of the viral transcript was performed with aTaqman assay designed to amplify a cMyc to WPRE fragment present in theSTEMCCA vector. Reactions were performed in triplicate using 1/20 of thecDNA obtained as described above. Gene expression levels were normalizedto-actin and relative quantification of expression was estimated usingthe comparative Ct method. For qRT-PCR analyses of changes in geneexpression in response to activin A stimulation, total RNA was purifiedwith RNeasy Mini kit (QIAGEN®) and treated with RNase-Free DNase I(QIAGEN®). One microgram of RNA was reverse-transcribed using TaqManReverse Transcription Reagents kit (APPLIED BIOSYSTEMS®) according tothe manufacturer's instructions. qPCR analyses of cDNAs was performed inan APPLIED BIOSYSTEMS® Sequence Detection System 7300 using thefollowing Taqman inventoried primers and probes (APPLIED BIOSYSTEMS®):Nanog (Mm02384862_g1), Rex1 (Mm01194089_g1), Brachyury (Mm00436877_m1),FoxA2 (Mm00839704_mH), Sox17 (Mm004883 63_m1), Gata4 (Mm00484689_m1),and Gata6 (Mm0080263 6_m1). Reactions were performed in duplicate using1/20 diluted cDNA obtained as described above. Gene expression levelswere normalized to 18S rRNA (4319413E) and relative expression of eachgene compared to undifferentiated ESC was quantified using the2^(−[delta][delta]Ct) method.

Bisulfite Sequencing

Bisulfite modification of genomic DNA (2 g) was carried out using theEpiTect Bisulfite Kit (QIAGEN®) following the protocol recommended bythe manufacturer. A region of the Nanog promoter was amplified byhot-start PCR as previously described in Takahashi K, and Yamanaka S.,Cell. 2006; 126:663-676. The Oct4 promoter w as amplified by nested-PCR.Initial PCR was done with the primers 5′-GTAAGTAAGAATTGAGGAGTGG-3′ (SEQ.ID. No. 43) and 5′-TCCAAACCCACCTAAAAACC-3′ (SEQ. ID. No. 44) under thefollowing conditions: 95° C. for 3 min, 30 cycles of 95° C. for 1 min,56° C. for 1 min and 72° C. for 1 min, and a final extension cycle at72° C. for 10 min. PCR products were purified with the QIAquick PCRPurification Kit (QIAGEN®) and 51 were used as a template for the secondPCR using the primers 5′-GATGGTTGAGTGGGTTGTAAGG-3′ (SEQ. ID. No. 45) and5′-CCAACCCTACTAACCCATCACC-3′(SEQ. ID. No. 46) and the conditionsdescribed above. Finally, purified PCR products were cloned into pGEM-Tvector (PROMEGA®) and sequenced with the T7 promoter primer.

Alkaline Phosphatase Staining and Immunofluorescence

Alkaline phosphatase staining was performed with the Vector RedSubstrate Kit (Vector Laboratories, Burlingame, Calif.) according to themanufacturer's protocol. For immunofluorescence, cells were fixed in 4%paraformaldehyde for 5 min, washed twice with PBS and incubated withmouse anti-SSEA-1 (Santa Cruz Biotechnology, Santa Cruz, Calif.) inPBS/5% goat serum for 30 min at 4° C. Secondary antibody staining wasperformed similarly using ALEXA® Fluor 568 goat anti-mouse IgM(INVITROGEN™, Inc.).

ES and iPS Cell Differentiation

Prior to differentiation all ESC and iPS cells were adapted toserum-free maintenance media (Gouon-Evans V, et al., Nat. Biotechnol.2006; 24:1402-1411) by culture expansion on mitomycin C-inactivatedmouse embryonic fibroblasts (MEFs). Maintenance media consisted of 50%Neurobasal medium (INVITROGEN™, Inc.) and 50% Dulbecco's Modified EagleMedium/F12 medium (INVITROGEN™, Inc.); with N2 and B27 supplements(INVITROGEN™, Inc.), 1% penicillin/streptomycin, 0.05% bovine serumalbumin, LIF (1000 U/ml; ESGRO; Chemicon; MILLIPORE®, Bedford, Mass.),10 ng/ml human BMP-4 (R&D Systems), and 1.5 10-4 M monothioglycerol(MTG) (SIGMA-ALDRICH®). Mouse ESC (129/Ola; containing GFP targeted tothe brachyury locus and hCD4 targeted to the Foxa2 locus) were thegenerous gift of Dr. Gordon Keller, Mount Sinai Medical Center, NewYork, N.Y. (Gouon-Evans V, et al., Nat. Biotechnol. 2006; 24:1402-1411;Gadue P, et al., Exp. Hematol. 2005; 33:955-964). Differentiation intoprimitive streak- and endoderm-like phenotypes was performed in serumfree media, as previously described by Keller and colleagues(Gouon-Evans V., et al. and Gadue P, et al. supra). Briefly, embryoidbodies were formed in suspension culture by plating ESC or iPS cells innon-adherent culture plates for 2 days in the absence of LIF. On day 2embryoid bodies were dispersed by trypsinization followed by replatingfor 3 more days in serum-free media with activin A (50 ng/ml; R&Dsystems, 338-AC). On day 5 embryoid bodies were dissociated withtrypsin/EDTA (2 min, 37° C.) and harvested for RNA extraction.

Teratoma Formation

One million iPS cells were injected subcutaneously into each flank ofrecipient NOD/SCID mice (Jackson Labs). The procedure was approved byParaffin sections of formalin-fixed teratoma specimens were prepared 3-5weeks after injection, and analysis of H and E stained tissue sectionswas performed for each specimen. All animal experiments were performedin accordance with Boston University Institutional Animal Care and UseCommittee (IACUC).

Southern Blot

Southern blot analysis using standard methods was performed on DNAdigested with BglII (New England Biolabs, MA) that cuts once in each ofthe two viral LTRs, in order to estimate the proviral copy number pergenome. In the Tet-STEMCCA vector a 8.3 kb band is expected. Because theEF1α promoter contains a BglII site, a smaller band of 6.7 Kb isexpected. Woodchuck Hepatitis virus post-transcriptional regulatoryelement (WPRE), a genomic fragment that enhances RNA export from thenucleus to the cytoplasm and therefore enhances viral titers duringproduction. A WPRE fragment that recognizes all our constructs was usedas a probe.

Generation of Chimeric Animals

Superovulation of C57BL/6J-Tyrc-2J donor females (The JacksonLaboratory) was induced by injection of 5 IU PMS followed by 5 IU HCG 48hours later. Female donors were immediately mated to stud males andchecked for vaginal plug formation the following day. Zygotes werecollected from the oviduct and cultured in KSOM media. Blastocysts wereidentified and injected with iPS cells before being surgicallytransferred to uteri of pseudopregnant females. Pregnant mice weresacrificed at day E11.5 and whole embryos were photographed with aninverted fluorescence microscope. Chimeric experiments were performed bythe Transgenic Center Core of Boston University School of Medicine.

Karyotyping and SKY Analysis

Metaphase spreads were prepared according to standard protocols (FrancoS, Mol. Cell. 2006; 21:201-214). Spectral karyotyping was performed witha mouse SKY paint kit (Applied Spectral Imaging Inc., Vista, Calif.)according to the manufacturer's instructions. Images were acquired withBX61 Microscope (Olympus, Tokyo) equipped with a motorized automaticstage, a cooled CCD camera and an interferometer (Applied SpectralImaging). 63× objective was used. Analysis was performed with the HiSKYand ScanView softwares (Applied Spectral Imaging).

Example 1 A Single Lentiviral Vector for the Expression of a Stem CellCassette

Previous studies have developed multicistronic lentiviral vectors basedon a combination of an IRES element and 2A peptide sequences (Szymczaket al., 2004, Nat Biotechnol 22, 589-594) to express multiple genessimultaneously from a single lentiviral vector (Chinnasamy et al., 2006,Virol. J. 3:14). Using a similar approach a single lentiviral transfergene plasmid was designed expressing a “STEM-Cell Cassette”, (hereafterpHAGE-STEMCCA). This cassette is comprised of a single multicistronicmRNA containing an IRES element separating two fusion cistrons. The twocistrons consist of Oct4 and Sox2 coding sequences fused to Klf4 andc-Myc, respectively, through the use of intervening sequences encoding‘self-cleaving’ 2A peptides (FIG. 1A). Two forms of pHAGE-STEMCCA weregenerated, wherein the multicistronic transcript is driven by either aconstitutive EF1α promoter or a doxycycline (dox)-inducible TetO-miniCMVpromoter. Both vectors resulted in the expression of all four individualproteins (OCT4, KLF4, SOX2, and c-MYC) as detected by western blotanalysis and immunohistochemistry (FIG. 1B. and data not shown). Theimmunofluorescence microscopy of MEFs infected 4 days earlier withpHAGE-EF1α-STEMCCA shows expression of all four transcription factors.Uninfected MEFs or secondary antibody only staining control showed nodetectable staining

The schematic plasmid map for pHAGE-EF1α-STEMCCA for constitutiveexpression of the four Oct4, Klf4, Sox2, and c-Myc transgenes is shownin FIG. 7 and the DNA sequence of the plasmid is SEQ. ID. No. 24.

The schematic plasmid map for pHAGE-Tet-STEMCCA for tet inducibleexpression of the four Oct4, Klf4, Sox2, and c-Myc transgenes is shownin FIG. 6 and the DNA sequence of the plasmid is SEQ. ID. No. 23.

Generation of iPS Cells with a Single Lentiviral Vector

Next, the capacity of pHAGE-STEMCCA to derive iPS clones from mouseembryonic or post-natal fibroblasts was assessed. As expected from thelarge size of the proviral genome of these plasmids (>9 Kb),pHAGE-STEMCCA viral titers (2−3×10⁸/ml) were lower than those obtainedusing monocistronic pHAGE plasmids (5×10⁹/ml). Nevertheless, mouseembryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) transducedwith the constitutive EF1α STEMCCA construct showed a dramatic change inmorphology already evident 6 days post-infection and formed coloniesthat were clonally expanded and displayed the typical morphology of EScell colonies (FIG. 2A).

For the generation of iPS cells with the dox-inducible construct, theTTFs that was transduced was from a Sox2-GFP Rosa26-M2rtTA doubleknock-in mouse in which the rtTA is constitutively expressed but theSox2-GFP allele is largely repressed. TTFs transduced with the induciblepHAGE-STEMCCA were exposed to doxycycline and changes in cell morphologywere evident 6-8 days post induction with colonies appearing at day12-14 (FIG. 2B). iPS colonies derived using either the constitutive(EF1α) or inducible (Tet) pHAGE-STEMCCA vector showed comparablealkaline phosphatase (AP) and SSEA1 staining as well as consistent andstrong GFP expression from the Sox2 locus, indicating reactivation of acrucial ES cell marker (FIGS. 2A and 2B). In addition, iPS clonesgenerated with either vector expressed a variety of other classic EScell marker genes (FIG. 2C) while these genes were not expressed infibroblasts prior to reprogramming. Furthermore, each iPS clone showedthe correct transmission of the full lentiviral vector genome asanalyzed by Southern blot and contained only 1-3 integrated viral copies(FIG. 2D). Expression of the STEMCCA mRNA transcript was determined invitro in iPS cells in the absence of doxycycline. As expected,transcript expression was found to be higher in iPS clones generatedwith the constitutively expressing vector compared to those generatedwith the dox-inducible vector (FIG. 5).

It was found that the pHAGE-STEMCCA lentiviral particle reprogramsfibroblasts with similar kinetics but higher efficiency compared toprior systems employing multiple vectors (Okita et al., 2007, Nature448, 313-317; Wernig et al., 2007, Nature 448, 318-324). Robustexpression of GFP from the Sox2 locus in TTFs following dox-induction ofreprogramming factors was detectable (by FACS and microscopy) at days8-9 of induction, similar to previous observations (Stadtfeld et al.,2008, Cell Stem Cell 2, 230-240). By day 16, approximately 15% of totalcells were GFP+ (FIG. 3). Approximately 50±8 (average+/−SD) GFP positivecolonies was obtained out of 100,000 TTFs exposed to pHAGE-STEMCCAlentiviruses. Taking into consideration the low viral transductionefficiency in our experiments (10-15%), which is likely due to the lowviral titers used, the effective reprogramming efficiency isapproximately 0.5%, 10-fold higher than that observed in prior reports(0.03-0.05%) (Okita et al., 2007, supra; Wernig et al., 2007 supra).

iPS Cells Generated with pHAGE-STEMCCA are Pluripotent

The capacity of iPS clones derived with constitutive and inducibleSTEMCCA vectors to differentiate into the three germ layers in teratomaswas assess. When injected into NOD/SCID mice, iPS cells derived fromboth vectors were capable of inducing teratoma formation with thegeneration of derivatives from all three germ layers (FIGS. 4A and 4B).Next, blastocyst injections were performed in order to further confirmthe pluripotency of iPS cells generated using pHAGE-STEMCCA. iPS cellsderived from the TTFs of Sox2-GFP Rosa26-M2rtTA mice using the induciblepHAGE-STEMCCA contributed to embryo development when injected intoblastocysts (FIG. 4C), as evidenced by Sox2-GFP expression in neuralcrest-derived tissues. Out of 14 implanted blastocysts, 12 developedinto mid-term embryos and 9 showed easily detectable GFP+iPS-derivedcells contributing to the chimeric embryos (FIG. 4C). These resultsindicate that iPS cells generated with a single viral vector arepluripotent.

The use of a single lentiviral vector for the derivation of iPS cellswill help reduce the variability in efficiency that has been observedbetween different laboratories, thus enabling more consistent geneticand biochemical characterizations of iPS cells and the reprogrammingprocess. From the safety perspective, we have shown that iPS cells canbe produced with minimal numbers of viral integrations, significantlyreducing the risks of insertional mutagenesis and viral reactivation.

Example 2

Reprogramming of somatic cells to a pluripotent state has been achievedby the introduction of four transcription factors, OCT4, KLF4, SOX2 andc-MYC using four independent retroviral vectors (Takahashi K. andYamanaka S., Cell. 2006, 126:663-676). Following the reproduction andextension of these studies in both murine and human cells (Okita K, etal., Nature. 2007, 448:313-317; Maherali N, et al., Cell Stem Cell.2007, 1:55-70; Wernig M, et al., Nature. 2007, 448:318-324; Takahashi K,et al., Cell. 2007, 131:861-872; Yu J, et al., Science. 2007;318:1917-1920), it is now widely accepted that iPS cells share many ofthe characteristics of embryonic stem cells (ESC), including geneexpression profiles, epigenetic signatures and pluripotency (Wernig M,et al., Nature. 2007, 448:318-324; Brambrink T, et al., Cell Stem Cell.2008; 2:151-159; Meissner A, et al., Nature, 2008, 454:766-70; MikkelsenT S, et al., Nature, 2007, 448:553-560; Stadtfeld M, et al., Cell StemCell. 2008, 2:230-240).

Since iPS cells can be generated from mature somatic cells, such as skinfibroblasts (Aasen T, et al., Nat. Biotechnol. 2008, 26:1276-1284; ParkI H, et al., Cell. 2008, 134(5):877-86; Dimos J T, et al., Science.2008, 321:1218-1221), enabling the derivation of patient-specific cellsand autologous tissues, it is often predicted that iPS cells will becomea powerful tool for biological research as well as a potent source forregenerative medicine. In order for this technology to become clinicallyrelevant, however, methods need to be developed that improve the safetyprofile of the technology while increasing the overall efficiency of theproduction of the cells. Several studies have demonstrated thatreactivation or sustained expression of reprogramming transgenes canresult in deleterious outcomes such as tumor formation (Maherali N, etal., Cell Stem Cell 2007, 1:55-70) or the disruption of pluripotency(Kopp J L, et al., Stem Cells. 2008, 26:903-911; Niwa H, et al., Nat.Genet. 2000, 24:372-376). Moreover, the study of the biology ofreprogramming and the ability to evaluate how closely iPS cells and ESCfunctionally resemble each other will greatly benefit from the use ofhomogeneous populations devoid of any residual transgene expression.

Recently, a series of studies have demonstrated proof of principle inthe generation of murine iPS cells without viral integrations (Okita K,et al., Science. 2008, 322:949-53; Stadtfeld M, et al., Science 2008,322:945-9). It should be noted, however, that the efficiency ofreprogramming in these studies was low and their application forderiving iPS cells from diverse, easily-accessible target somatic cellsappears to be limited.

Similarly, transposon-mediated reprogramming, albeit at low efficiency,was able to generate murine iPS cells free of exogenous factors (WoltjenK, et al., Nature. 2009, 458:766-70). The potential toxicity associatedwith the use of the transposon/transposase system in iPS cells, however,remains to be studied (Geurts A M, et al., PLoS Genet. 200, 2:e56; WangW, et al., Proc. Natl. Acad. Sci. U.S.A. 2008, 105:9290-9295). Mostrecently, Jaenisch and colleagues demonstrated Cre-mediated excision ofmultiple integrated reprogramming lentiviral vectors in human iPS cellsfollowing the completion of reprogramming (Soldner F, et al., Cell.2009, 136:964-977). This important advance demonstrated that prior toexcision the transcriptome of iPS cells differs slightly from controlESC. To date it remains unclear whether these subtle differences inglobal gene expression between iPS cells and ESC are accompanied bysignificant functional differences. Moreover, if indeed removal oftransgenes is necessary for proper functioning of iPS cells, methodsthat are simple, efficient, and involve minimal screening strategies forderiving ‘transgene-free’ iPS cells represent important advances for theclinical translation of this technology.

As described herein in Example 1 is the use of a single lentiviral ‘stemcell cassette’ (STEMCCA) for the efficient generation of iPS cells frompost-natal fibroblasts (Sommer C A, et al., Stem Cells. 2009;27:543-549). Importantly, the use of a single polycistronic vector,expressing Oct4, Klf4, Sox2, and c-Myc, allowed the inventors to obtainiPS cell clones with a single integration. Here the STEMCCA vector wasadapt to derive iPS cells free of exogenous reprogramming transgenes.Using a single integrated copy of this polycistronic vector, encodingeither 3 or 4 reprogramming factors flanked by loxP sites, efficientreprogramming of post-natal fibroblasts was accomplish, followed byhighly efficient Cre-mediated excision of the vector. A directcomparison of iPS cell clones before and after excision reveals thatremoval of the reprogramming vector markedly improves the developmentalpotential and differentiation capacity of iPS cells.

Cre-Mediated Excision of a loxP-Containing Polycistronic ReprogrammingVector Allows the Derivation of ‘Transgene-Free’ iPS Cells

In order to develop a simplified method for the derivation oftransgene-free iPS cells, a vector system that would result in efficientreprogramming with a single reagent, without the need for concurrentadditional vectors, transgenes, or chemical exposures was developed andutilized. Hence, in contrast to other studies in which an induciblesystem was used (Stadtfeld M, et al., Cell Stem Cell. 2008; 2:230-240;Soldner F, et al., Cell. 2009; 136:964-977; Wernig M, et al., Nat.Biotechnol. 2008; 26:916-924) constitutively expressed versions of thelentiviral STEMCCA vector under regulatory control of a human EF1promoter was selected for this example. This single reagentaccomplishes efficient and reliable reprogramming of post-natal cells byexpressing four factors (OCT4, KLF4, SOX2, and c-MYC) and obviates theneed for additional genetic modification since the transactivator (i.e.rtTA) is not required to induce expression of the reprogrammingcassette. Because previous studies have shown that iPS cells can bederived without the presence of exogenous cMyc (Nakagawa M, et al., Nat.Biotechnol. 2008; 26:101-106), a modified 3 factor STEMCCA vector bysubstituting cMyc with the coding sequence of the red fluorochromemCherry was also developed. This modified vector, hereafter namedSTEMCCA-RedLight, constitutively expresses mCherry as well as the 3reprogramming factors, Oct4, Klf4 and Sox2 from a single polycistronicmRNA, thus allowing monitoring of STEMCCA gene expression in livingcells.

In order to allow for excision of the 3 factor or 4 factor STEMCCAvectors, we first introduced a loxP site in the deleted U3 (dU3) regionof each lentiviral vector's 3′LTR (Sommer C A, et al., Stem Cells. 2009;27:543-549) (FIG. 8A). During the normal reverse transcription cycle ofthe virus before integration, the U3 region is copied to the 5′ LTR ofthe proviral genome, creating a loxP-flanked or ‘foxed’ version of theSTEMCCA vector that integrates into the host chromosome. These floxedSTEMCCA vectors (hereafter STEMCCA-loxP and STEMCCA-loxP-RedLight) wereused to generate iPS cells from tail tip fibroblasts (TTFs) of Sox2-GFPknock in mice as previously described herein and in Sommer C A, et al.,supra. The introduction of loxP sites did not affect viral titers (datanot shown), and the STEMCCA-loxP vector was able to generate iPS cellcolonies with the same kinetics and the same reprogramming efficiency(˜0.5%) as demonstrated previously using STEMCCA herein and in Sommer CA, et al., supra. Initial selection of iPS colonies generated with thisvector was based solely on morphological criteria and 20 out of 24 (83%)picked colonies selected on this basis resulted in Sox2-GFP expressingcell lines after expansion. As expected 3 factor reprogramming usingSTEMCCA-loxP-RedLight was slower and less efficient than 4 factorreprogramming. Sox2-GFP+ colonies appeared only 25-30 days aftertransduction with STEMCCA-loxP-RedLight (compared to 15-20 days whenusing the four factors vector), and overall reprogramming efficiency was0.01%, or 50 fold lower than that observed with STEMCCA-loxP.Importantly, persistent expression of the polycistronicSTEMCCA-loxP-RedLight vector, driven by the constitutively active EF1promoter could be readily visualized by red fluorescence microscopyduring reprogramming and was maintained in picked Sox2-GFP+iPS clonesafter the completion of reprogramming (data not shown).

Next, Sox2-GFP expressing clones were screened by Southern blot todetermine the number of viral integrations, as a first step to pursuevector excision. gDNA was digested with BamHI to expose each individualviral integration. Both, SEFL1 and SEFL2 clones displayed a singleintegration that is not detected after exposure to Cre (SEFL1-Cre andSEFL2-Cre). Three of 9 screened clones generated with STEMCCA-loxP and 3of 7 clones generated with STEMCCA

loxP-RedLight showed single copy integration (FIG. 12). In order toexcise the single integrated copy of each floxed vector, the clones wereexposed to an adenoviral vector (Adeno-Cre) to achieve transientexpression of Cre recombinase. Adeno-Cre mediated recombination wasemployed rather than electroporation of Cre expressing plasmids based onscreening studies revealing superior transfection efficiencies of ESC oriPS cells using adenoviral vectors vs. plasmid electroporation (90-100%transfection efficiency vs. 0.5-1%, respectively, data not shown).Adeno-Cre infection of all single integrant iPS cell lines (n=3)resulted in successful Cre-mediated excision of STEMCCA-loxP in 5 out of5 subclones of each cell line, as evidenced by PCR of gDNA (FIG. 13). Inaddition, Southern blot analysis confirmed the absence of integratedvector using probes against the WPRE sequence of the STEMCCA vector(FIG. 8C) as well as against the individual reprogramming genes (FIG. 8Cand data not shown). PCR screening using oligos specific to Cre was usedto confirm that, as expected, no integration of the Adeno-Cre vector hadoccurred (data not shown). As e xpected, following excision of thereprogramming cassette and culture expansion of excised iPS cellsubclones, the STEMCCA transcript was undetectable as evidenced byRT-PCR (FIG. 8D) and qRT-PCR (FIG. 14), in contrast to the pre-excisionparental clones. In iPS cells generated with STEMCCA-loxP-RedLight,disappearance of the mCherry reporter was used to precisely monitor andquantify STEMCCA excision efficiency by fluorescence microscopy (datanot shown) and FACS (FIG. 8B). These iPS cells co-expressed mCherry andSox2-GFP, however, after Adeno-Cre infection, 96 out of 100 coloniesexpressed only GFP but not mCherry, suggesting an excision efficiency ofalmost 100%.

Similar results were obtained when probing for Klf4 (FIG. 8C). In thiscase, the endogenous gene was evident in all clones (closed arrowhead)while the STEMCCA encoded Klf4 is not present following Cre excision(open arrowhead). Gemonic DNA was digested with BglII to obtain a bandof 6.7 Kb that confirms appropriate viral transmission. For a control,STEMCCA-loxP plasmid DNA representing 2.5 copies of the insert wasdigested with BglII. A single band corresponding to an integration ofthe correct size was observed in SEFL1 and SEFL2 clones and was notpresent following Cre treatment.

iPS Cells Display Stable Growth Characteristics and Stem Cell MarkerGene Expression after Excision of Reprogramming Transgenes

For further study of iPS cells following excision of the reprogrammingtransgenes, two subclones generated after excision of STEMCCA-loxP wereselected and named SEFL1-Cre and SEFL2-Cre, based on their origin fromthe parental SEFL1 and SEFL2 clones, respectively (FIG. 8C). As shown inFIGS. 9A and 9B, after STEMCCA excision iPS cells maintained expressionof alkaline phosphatase (AP), SSEA1, Sox2-GFP and a variety of stem cellmarkers as evidenced by RT-PCR. In addition, the methylation status ofthe Oct4 and Nanog proximal promoter regions was analyzed by bisulphitesequencing (FIG. 9C). Both before and after STEMCCA-loxP excision,established iPS cell clones exhibited unmethylated CpG islands at thesekey loci, in contrast to parental fibroblasts. Importantly, iPS cellexpansion appeared to be stable following excision of the STEMCCA-loxPas evidenced by the ability of the cells to be maintained in theundifferentiated state in culture for at least 20 passages.

Excision of Reprogramming Transgenes Facilitates the DevelopmentalCapacity of iPS Cells

As previously reported, the iPS cells generated with the constitutiveSTEMCCA vector were able to differentiate into all three germ layers interatoma assays, despite the residual expression of exogenousreprogramming genes (in Example 1 and Sommer C A, et al., Stem Cells.2009; 27:543-549). Indeed, these results were confirmed using the SEFL1or SEFL2 clones regardless of whether these clones were tested before orafter excision of the STEMCCA-loxP, SEFL1 and SEFL2 iPS cell linesreadily gave rise to differentiated cells of all three primary germlayers in teratoma assays (FIG. 10A).

Constitutive expression of STEMCCA, however, did appear to adverselyaffect the in vivo developmental potential of iPS cells aftertransplantation into mouse blastocysts. As shown in FIG. 10B, iPS cellsgenerated with the constitutive STEMCCA vector were injected into 15blastocysts followed by midgestation (E11.5) harvest. These injectionsyielded only 5 embryos of which 3 were chimeric. All 3 chimeric embryos,however, displayed severe morphological abnormalities. In addition,following four independent attempts we were unable to obtain livechimeras when using three different iPS cell clones containing theconstitutive STEMCCA or STEMCCA-loxP vector. In marked contrast,following Cre-mediated excision of STEMCCA-loxP, iPS cells injected into14 blastocysts yielded 11 midgestation embryos of which 7 were chimericwith 5/7 displaying normal developmental morphology (FIG. 10C).Moreover, we were able to obtain live chimeric mice from blastocystsinjected with iPS cells after excision of STEMCCA-loxP (FIG. 10D).

While these results indicated that sustained expression of thereprogramming genes may affect the ability of iPS cells to undergoappropriate embryonic development in chimeric embryos, it is not clearwhether the morphological defects in the chimeric embryos resulteddirectly from failure of the injected iPS cells to differentiate. Indeedthe seemingly intact ability of these same iPS clones to undergotri-lineage differentiation in teratoma assays indicates they retainsome capacity to respond to differentiation cues such as soluble growthfactors. Hence, in order to more fully evaluate the effects of residualSTEMCCA expression on the capacity of iPS cells to differentiate inresponse to normal developmental cues active in the early embryo, iPScells in culture were stimulated with activin A (hereafter activin), aprotein known to mimic embryonic nodal/TGF signaling. Activin A has beenshown to induce ESC in vitro to differentiate sequentially intoprimitive streak-like cells followed by definitive endoderm (Gouon-EvansV, et al., Nat. Biotechnol. 2006; 24:1402-1411; Gadue P, et al., Exp.Hematol. 2005; 33:955-964; Kubo A, et al., Development. 2004;131:1651-1662).

iPS cells before (SEFL1 and SEFL2) and after (SEFL1-Cre and SEFL2-Cre)excision of STEMCCA-loxP are compared to an ESC clone. mRNA extracted onday 0 (−) and day 5 (+) of activin stimulation was used as the templatefor RT-PCR. Several genes characteristic of ESC, i.e. Rex1, Sox2, Esg1and Nanog, as well as primitive streak (Brachyury: Bry) and earlyendoderm (FoxA2) markers are depicted. Note lack of induction of theneuroectoderm marker Pax6. Samples prepared without RT were used as thenegative control (—RT).

Following Cre-mediated excision of STEMCCA-loxP, ‘transgene-free’versions of each iPS cell clone were compared to their parental clonesin terms of endodermal potential in vitro in response to activin A. Intwo independent experiments, analyzed by either conventional orquantitative RT-PCR, each constitutive, integrated STEMCCA-loxP cloneshowed diminished potential to upregulate endodermal markers in responseto activin A in vitro (FIGS. 11A and 11B). Although all clones were ableto form embryoid bodies in culture, STEMCCA-loxP containing clonesshowed little potential to upregulate key endodermal transcriptionfactors, such as brachyury, FOXA2, SOX17, GATA4, and GATA6. Furthermore,these clones showed persistent expression of the pluripotent markersRex1 and Esg1 after activin A stimulation (FIG. 11A). In contrast,excision of STEMCCA-loxP from these clones improved the capacity of eachclone to upregulate endodermal transcription factors and downregulatepluripotent loci in response to activin (FIGS. 11A and 11B). Takentogether these results underscore the importance of obtaining iPS cellsfree of exogenous transgenes, not only as a means to improve the safetyprofile of iPS cells but also to enable their appropriatedifferentiation potential.

iPS Cells Before and after Excision of Reprogramming Transgenes DisplayFrequent Trisomy of Chromosome 8

The possibility that Cre recombination in iPS cells might causechromosomal instability, such as translocation events, was considered.In doing so, karyotyping by light microscopy was performed as well asspectral karyotyping analyses (SKY) on several iPS cell lines before andafter Cre-mediated excision of STEMCCA-loxP. Importantly, notranslocation events were detected in any of the clones analyzedstrongly suggesting that removal of the single integration byCre-excision has no deleterious effects on the genome of transgene-freeiPS cells. However, it was noted that the majority of iPS cells fromthree out of six clones displayed frequent trisomy of chromosome 8 (datanot shown). This chromosomal abnormality appeared to be present prior toCre-mediated excision of STEMCCA-loxP, but was not present in theparental fibroblast cell line prior to reprogramming (data not shown).In some cells analyzed by SKY (data not shown) an apparently normalnumber of 40 mouse chromosomes masked the loss of the Y chromosome incombination with trisomy of chromosome 8. These chromosomalabnormalities have been well described in murine ESC with markedlyincreased frequencies with increased passage number (Ensenat-Waser, etal., In Vitro Cell Dev. Biol. Anim. 2006, 42:1 15-123; Liu X, et al.,Dev. Dyn. 1997, 209:85-91). Trisomy 8 is the most common chromosomalabnormality found in murine ESC and is known to increase growth kineticsand preclude germ line transmission (Liu X, et al., 1997, supra). Thehigh frequency of trisomy 8 abnormalities observed in our iPS cellclones was already present by passage 8 and likely favored theiroutgrowth during culture, as has been described in ESC.

The present disclosure demonstrate one approach for deriving‘transgene-free’ iPS cells using a floxed single copy of a polycistronicreprogramming vector. This method allows head-to-head comparisons of thefunctional capacity of iPS cells before and after excision ofreprogramming transgenes. The findings herein emphasize the importanceof vector excision prior to directed differentiation of iPS cells inculture, if the goal is to adapt differentiation culture conditionsoriginally developed in ESC culture systems.

This is the first evaluation of the in vitro endodermal potential of iPScells in serum-free defined culture conditions. In order to generatedifferentiated precursor cells for modeling or treating human diseases,it is crucial to first show that iPS cells can be directed in culture torecapitulate the sequence of developmental milestones involved in germlayer formation and differentiation. The results indicate that transgenefree iPS cells upregulate an endodermal differentiation program inresponse to the same serum-free culture conditions previously employedto derive definitive endoderm from ESC (Gouon-Evans V, et al., Nat.Biotechnol. 2006). Although persistent expression of reprogrammingfactors diminished the response of iPS cells to activin in vitro, theprecise mechanism for this effect is not clear. During differentiationstem cells exhibit downregulation of loci encoding pluripotentregulators accompanied by activation of master transcriptionalregulators of differentiation. The results herein indicate thatpersistent expression of reprogramming factors interferes withappropriate downregulation of pluripotent loci, such as Rex1 and Esg1(FIG. 11). Whether persistence of a general pluripotent gene program, orany particular specific reprogramming factor directly resulted infailure to upregulate the endodermal transcriptional regulators FOXA2,SOX17, and GATA4/6 will require further investigation. Recent worksuggests that c-MYC over-expression during reprogramming leads todownregulation of somatic differentiation gene programs, whereas OCT4,KLF4, and SOX2 over-expression triggers activation of pluripotency(Sridharan R, Cell. 2009, 136:364-377). It is conceivable that similarmechanisms account for the present findings when reprogrammed cells arethen stimulated to differentiate.

The development of methods to derive iPS cells free of exogenous geneticmaterial is particularly important if iPS cells are to be employed forregenerative therapies in human trials. By excising all reprogrammingtransgenes the disclosed approach herein eliminates the risk ofoncogenic transgene reactivation following transplantation ofiPS-derived cells. In addition, by excision of only a single vectorcopy, the disclosed approach herein minimizes the risk of chromosomaltranslocations, an advance over prior methods for Cre-mediated excisionof multiple copies of individual reprogramming lentiviral vectors(Soldner F, et al., Cell. 2009, 136:964-977).

It should be noted that a number of technical hurdles complicate the useof Cre-mediated excision of DNA sequences from stem cells, potentiallylimiting the application of these methods to easily generate iPS cellsfree of floxed transgenes. First, delivery of Cre to ESC or iPS cellshas been previously noted to be inefficient; second, screening methodsto detect successful Cre-recombination may be cumbersome; and third,clumping of cells after delivery of Cre. The mCherry-containingSTEMCCA-loxP-RedLight vector described herein can be particularlyhelpful in surmounting these hurdles, since this single reagentaccomplishes effective ‘3 factor reprogramming’, and the resultsdemonstrate that Cre-recombination efficiency and excision ofreprogramming transgenes from the resulting iPS cells can be readilyvisualized and monitored in individual living cells and colonies inculture. Indeed, it was found that adenoviral delivery of Cre as well asCre recombination to be highly efficient in iPS cells using monitoringof mCherry disappearance to optimize our transgene excision methodology.Furthermore, FACS sorting based on mCherry expression may be easilyemployed by those investigators wishing to rapidly separate excised fromunexcised iPS cells.

It was also found that trisomy of chromosome 8 was frequent in these iPScell lines independent of Cre-mediated excision of reprogrammingtransgenes. This trisomy is common in ESC but has not been reportedpreviously in iPS cells. While this observation further emphasizes thesimilarity between iPS cells and ESC, it is not yet known whether theoverexpression of reprogramming factors facilitates this chromosomalabnormality. In ESC, frequency of this trisomy increases with passagenumber, and it is possible that the several passages required togenerate stable iPS cells during the gradual reprogramming process allowfor more prevalent trisomies in iPS cells than has been previouslyappreciated. The additional passages required to screen for iPS cellclones with single vector copies, perform vector excision, re-screen forexcision, and characterize the transgene-free clones may be particularlyproblematic in terms of chromosomal instability. For this reason,methods to reprogram cells, maintain chromosomal stability, and exciseresidual transgenes with the highest efficiency and the lowest possiblepassage number will be important if iPS cells are to be translated forhuman therapies. Therefore proposed herein is the use of theSTEMCCA-loxP-RedLight vector as a useful tool for accomplishing thisgoal, since excision of the mCherry ‘RedLight’ serves as a simpleindicator, requiring minimal screening, for the generation oftransgene-free iPS cells.

The contents of all references cited throughout this application, aswell as the figures and table are incorporated herein by reference.

What is claimed:
 1. A lentiviral vector comprising a nucleic acidsequence operatively linked to a promoter, wherein the nucleic acidsequences comprises: a. a ‘self-cleaving’ 2A peptide; b. an internalribosome entry site (IRES); and c. a first gene, a second gene, andthird gene selected from the group of genes consisting of Oct4, Klf4 andSox2; wherein the first gene, second gene and third gene can be in anyorder and wherein the ‘self-cleaving’ 2A peptide is positioned betweenthe first gene and second gene, and wherein the IRES is positionedbetween the second and third genes, and wherein the nucleic acidsequence encoding the first gene, second gene and third gene, the‘self-cleaving’ 2A peptide and the IRES are transcribed from thepromoter as a multi-cistronic RNA.
 2. The lentiviral vector particle ofclaim 1 further comprising a marker gene, wherein the marker geneencodes an optically visible protein or an enzyme.
 3. The lentiviralvector particle of claim 1, wherein the ‘self-cleaving” 2A peptide isselected from the group consisting of F2A, E2A, T2A and P2A.
 4. Thelentiviral vector particle of claim 1, wherein the order of the first,second and third genes is Oct4, Klf4 and Sox2.
 5. The lentiviral vectorparticle of claim 1 further comprises a Cre-LoxP excision sequence.
 6. Amethod of producing an induced pluripotent stem (iPS) cell comprisingtransducing a somatic cell with the lentiviral vector of claim
 1. 7. Themethod of claim 6, wherein the somatic cell is a mammalian cell.
 8. Themethod of claim 7, wherein the mammalian cell is a human cell.
 9. A cellcomprising the lentiviral vector of claim
 1. 10. The cell of claim 9,wherein the cell is a mammalian cell.
 11. The cell of claim 11, whereinthe mammalian cell is a human cell.